Brief

Report

Eye

Movements

During

Visual

Search

Tasks

Indicate

Enhanced

Stimulus

Discriminability

in

Subjects

with

PDD

Subjects

with

PDD

excel

on

certain

visuo-spatial

tasks

amongst

which

visual

search

tasks

and

this

has

been

attributed

to

enhanced

perceptual

discrimination

However

an

alternative

explanation

is

that

subjects

with

PDD

show

a

different

more

effective

search

strategy

The

present

study

aimed

to

test

both

hypotheses

by

measuring

eye

movements

during

visual

search

tasks

in

high

functioning

adult

men

with

PDD

and

a

control

group

Subjects

with

PDD

were

significantly

faster

than

controls

in

these

tasks

replicating

earlier

findings

in

children

Eye

movement

data

showed

that

subjects

with

PDD

made

fewer

eye

movements

than

controls

No

evidence

was

found

for

a

different

search

strategy

between

the

groups

The

data

indicate

an

enhanced

ability

to

discriminate

between

stimulus

elements

in

PDD

Introduction

While

most

diagnostic

criteria

of

Pervasive

Developmental

Disorder

-LRB-

PDD

-RRB-

are

characterised

by

impairments

mostly

with

respect

to

social

and

communicational

abilities

subjects

with

PDD

excel

on

certain

visuo-spatial

tasks

that

supposedly

reflect

superior

processing

of

fine

detail

-LRB-

see

Dakin

Frith

for

a

review

-RRB-

An

especially

robust

finding

seems

to

be

the

behavior

of

subjects

with

PDD

on

visual

search

tasks

-LRB-

Plaisted

O'Riordan

Baron-Cohen

Plaisted

Saksida

Alcantara

Weisblatt

O'Riordan

Plaisted

Driver

Baron-Cohen

-RRB-

In

such

tasks

subjects

are

required

to

detect

a

target

in

a

display

containing

a

variable

number

of

distracters

If

the

task

difficulty

is

increased

by

adding

distracters

usually

RTs

increase

In

studies

on

PDD

two

search

task

versions

that

differed

in

difficulty

have

been

used

In

both

tasks

children

with

PDD

showed

shorter

RTs

as

compared

with

normally

developing

controls

-LRB-

Plaisted

et

al.

Plaisted

et

al.

O'Riordan

et

al.

-RRB-

The

reason

for

this

superior

performance

is

not

clear

but

has

been

related

to

enhanced

ability

to

discriminate

between

stimulus

elements

in

subjects

with

PDD

-LRB-

O'Riordan

Plaisted

-RRB-

an

explanation

that

has

also

been

proposed

for

the

superior

performance

of

subjects

with

autism

on

other

visuo-spatial

tasks

-LRB-

Plaisted

et

al.

see

also

Bertone

Mottron

Jelenic

Faubert

-RRB-

So

far

however

there

have

been

no

studies

that

have

validated

the

claim

for

enhanced

stimulus

discrimination

as

an

explanation

for

superior

performance

in

search

tasks

in

PDD

more

directly

It

is

well-known

that

stimulus

discriminability

affects

eye

movement

parameters

especially

number

of

fixations

and

fixation

duration

-LRB-

Hooge

Erkelens

-RRB-

Therefore

studying

eye

movements

during

search

tasks

in

subjects

with

PDD

is

a

first

step

to

gain

more

insight

into

the

neurocognitive

mechanisms

of

atypical

visuo-spatial

processing

in

this

group

If

increased

stimulus

discriminability

is

indeed

the

underlying

factor

for

superior

performance

of

subjects

with

PDD

it

is

expected

that

they

need

fewer

and/or

shorter

fixations

to

identify

the

target

Also

eye

movement

parameters

can

be

used

to

test

alternative

hypotheses

for

shorter

reaction

times

in

search

tasks

in

PDD

More

specifically

it

has

been

argued

that

-LRB-

healthy

-RRB-

subjects

in

a

visual

search

task

have

a

tendency

to

move

their

eyes

even

though

in

some

situations

it

would

be

a

better

strategy

not

to

do

so

since

longer

fixations

allow

better

extraction

of

-LRB-

peripheral

-RRB-

information

-LRB-

Hooge

Erkelens

see

also

Rayner

-RRB-

It

is

well

possible

that

individuals

with

PDD

use

a

different

search

strategy

in

which

they

show

longer

but

less

fixations

than

controls

The

first

aim

of

the

present

study

was

to

use

eye

movement

parameters

to

test

the

two

hypotheses

described

above

for

superior

visual

search

in

subjects

with

PDD

A

second

aim

was

to

replicate

the

findings

of

Plaisted

et

al.

-LRB-

-RRB-

and

O'Riordan

and

Plaisted

-LRB-

-RRB-

in

high

functioning

adults

with

PDD

For

these

reasons

we

used

the

same

search

tasks

as

described

in

O'Riordan

et

al.

-LRB-

second

experiment

-RRB-

in

a

group

of

high

functioning

adults

with

PDD

matched

on

gender

IQ

and

age

to

a

control

group

Methods

Participants

Two

groups

of

young

male

adults

participated

a

group

of

eight

high

functioning

individuals

with

PDD

and

a

group

of

eight

normal

control

individuals

The

clinical

subjects

were

recruited

via

the

Department

of

Child

and

Adolescent

Psychiatry

at

the

Utrecht

University

Hospital

The

study

was

described

to

the

subjects

and

written

informed

consent

was

obtained

All

subjects

were

administered

the

Wechsler

Intelligence

Scale

revised

Dutch

edition

-LRB-

WAIS

-RRB-

Diagnoses

of

either

Autistic

Disorder

or

Asperger

Syndrome

were

made

by

a

child

psychiatrist

and

based

on

DSM-IV

criteria

The

parents

of

all

subjects

with

PDD

were

administered

the

Autism

Diagnostic

Interview

Revised

-LRB-

ADI-R

-RRB-

-LRB-

Lord

Pickles

McLennan

Rutter

Lord

Rutter

LeCouteur

-RRB-

Due

to

technical

problems

eye-movement

data

of

one

subject

with

PDD

and

one

control

subject

were

lost

The

individual

ADI

scores

of

the

remaining

seven

PDD

subjects

can

be

found

in

Table

Unpaired

t-tests

revealed

that

there

were

no

significant

differences

between

the

remaining

seven

PDD

and

seven

control

subjects

with

respect

to

either

age

TIQ

VIQ

or

PIQ

-LRB-

see

Table

-RRB-

of

the

participants

showed

any

visual

or

oculomotor

pathology

other

than

refraction

anomalies

Subjects

were

allowed

to

wear

their

contact

lenses

or

glasses

Table

scores

on

the

four

domains

for

individual

PDD

subjectsADI

scores

individual

subjectsSocial

interaction

-LRB-

cutoff

-RRB-

Communication

-LRB-

cutoff

-RRB-

Stereotypy

-LRB-

cutoff

-RRB-

Age

of

onset

-LRB-

cutoff

-RRB-

age

and

IQ

characteristics

of

the

control

and

PDD

Set

up

and

Data

Analysis

Subjects

sat

in

front

of

a

LaCie

Blue

Electron

lll

Screen

-LRB-

m

m

pixels

at

Hz

-RRB-

at

a

distance

of

m.

Stimuli

were

generated

by

a

Apple

PowerMac

DP

using

a

Matlab

program

based

on

routines

taken

from

the

Psychophysics

Toolbox

-LRB-

Brainard

Pelli

-RRB-

and

the

EyeLink

Toolbox

-LRB-

Cornelissen

Peters

Palmer

-RRB-

Movements

of

the

left

eye

were

measured

at

Hz

with

the

EyeLink

eye

monitor

Head

movements

were

prevented

by

the

use

of

a

chinrest

Data

were

stored

on

disk

and

were

analysed

off-line

by

a

self-written

Matlab

program

The

velocity

signal

of

eye

movements

was

searched

for

peak

velocities

above

s.

Each

peak

-LRB-

in

the

velocity

signal

-RRB-

was

considered

a

potential

indicator

of

the

presence

of

a

saccade

The

exact

onset

of

the

saccade

was

determined

by

going

backward

in

time

to

the

point

where

the

velocity

signal

dropped

below

the

average

velocity

plus

two

standard

deviations

during

the

stable

fixation

period

before

the

saccade

The

exact

offset

of

the

saccade

was

determined

by

going

forward

in

time

to

the

point

where

the

velocity

signal

dropped

below

the

average

velocity

plus

two

standard

deviations

during

the

stable

fixation

period

after

the

saccade

This

method

was

adopted

from

Van

der

Steen

and

Bruno

-LRB-

-RRB-

This

procedure

was

followed

by

rejection/acceptation

based

on

minimum

saccade

duration

of

ms

and

minimum

amplitude

of

When

a

saccade

was

removed

fixation

time

before

and

after

this

saccade

and

the

duration

of

the

saccade

were

added

together

Stimulus

and

Task

Two

tasks

were

tested

in

separate

sessions

for

each

participant

with

the

order

counterbalanced

within

participant

groups

Both

tasks

contained

tilted

as

well

as

vertical

line

elements

The

tasks

only

differed

in

which

of

the

two

elements

was

target

or

distracter

Since

an

earlier

study

has

shown

a

difference

in

task

difficulty

depending

on

target

orientation

-LRB-

O'Riordan

et

al.

-RRB-

the

condition

including

a

vertical

target

among

tilted

distracters

will

be

referred

to

as

the

easy

condition

and

the

condition

with

a

tilted

target

-LRB-

among

vertical

distracters

-RRB-

as

the

hard

condition

Each

task

contained

two

crossed

factors

set

size

-LRB-

or

items

-RRB-

and

probe

target

present

or

target

absent

There

were

trials

at

each

unique

combination

of

factors

yielding

a

session

of

trials

per

task

The

sequence

of

different

tasks

was

randomised

within

each

session

In

each

session

the

participant

was

informed

about

the

nature

of

the

target

-LRB-

either

vertical

or

tilted

-RRB-

but

did

not

know

when

a

target

would

be

present

or

absent

or

what

the

display

size

would

be

on

any

trial

The

participants

performed

a

binary

choice

RT

task

indicating

whether

a

target

was

present

or

absent

by

a

button

press

on

each

trial

Procedure

The

order

in

which

the

two

conditions

were

presented

was

counterbalanced

Prior

to

each

task

participants

were

given

practice

trials

Participants

were

instructed

to

respond

as

quickly

and

accurate

as

possible

by

pressing

the

five

key

on

the

numeric

pad

when

the

target

was

present

and

the

four

key

when

the

target

was

absent

Thereafter

the

eye

link

camera

was

attached

to

the

head

and

calibrated

On

each

trial

the

sequence

of

events

was

as

follows

a

fixation

cross

was

presented

The

participant

had

to

fixate

at

the

cross

and

press

the

space

bar

subsequently

Fixation

of

the

fixation

cross

was

used

for

on-line

drift

correction

of

the

eye

tracker

Then

the

search

display

appeared

and

remained

visible

until

the

participant

responded

After

the

response

the

next

trial

started

Results

To

assure

that

a

possible

superior

task

performance

of

the

individuals

with

PDD

was

not

due

to

a

difference

in

detection

criteria

-LRB-

e.g.

if

speeded

reaction

time

is

accompanied

by

a

decrease

in

number

of

correct

answers

-RRB-

we

performed

an

independent

t-test

on

the

error

rates

of

both

groups

No

significant

difference

in

error

rate

between

the

groups

was

found

One

of

the

subjects

in

the

PDD

group

showed

a

high

number

of

errors

and

a

deviant

pattern

in

reaction

time

data

and

was

excluded

from

further

analysis

The

results

described

below

are

therefore

based

on

six

subjects

with

PDD

and

seven

controls

The

mean

RT

first

fixation

times

remaining

fixation

times

and

number

of

fixations

data

were

analysed

using

a

repeated

measures

ANOVA

with

one

between

subjects

factor

of

group

-LRB-

control

or

autistic

-RRB-

and

three

within

subject

factors

condition

-LRB-

condition

tilted

target

or

condition

vertical

target

-RRB-

probe

-LRB-

target

present

or

target

absent

-RRB-

and

set

size

-LRB-

or

items

-RRB-

Analyses

of

the

first

and

remaining

fixations

were

done

separately

because

there

is

evidence

that

they

belong

to

different

distributions

-LRB-

Hooge

Erkelens

-RRB-

Unless

otherwise

stated

a

significance

level

of

p

was

adopted

for

all

statistical

comparisons

Only

main

effects

or

interactions

with

Group

will

be

considered

RT

Analysis

The

analysis

of

variance

revealed

a

main

effect

of

Group

F

-LRB-

-RRB-

p

reflecting

the

fact

that

individuals

with

PDD

were

significantly

faster

overall

than

the

control

individuals

-LRB-

vs.

ms

-RRB-

A

significant

interaction

Group

Display

was

found

-LRB-

F

-LRB-

-RRB-

p

-RRB-

as

well

as

an

interaction

of

Group

Display

Probe

-LRB-

F

-LRB-

-RRB-

p

-RRB-

Partial

analyses

showed

that

the

Groups

difference

was

significant

at

all

set

sizes

but

that

the

effects

were

largest

for

set

size

-LRB-

all

p

-RRB-

-LRB-

resp.

F

-LRB-

-RRB-

-RRB-

The

interaction

Group

Probe

was

marginally

significant

for

set

size

only

-LRB-

F

-LRB-

-RRB-

p

-RRB-

indicating

that

at

this

set

size

the

Groups

difference

was

largest

if

the

target

was

absent

-LRB-

See

Fig.

-RRB-

Fig.

and

standard

errors

of

the

reaction

times

for

each

group

in

trials

with

-LRB-

left

panel

-RRB-

and

without

a

target

-LRB-

right

panel

-RRB-

for

the

easy

and

hard

condition

Eye

Movement

Analysis

The

analysis

of

number

of

fixations

revealed

a

significant

effect

of

group

-LRB-

F

-LRB-

-RRB-

p

-RRB-

The

individuals

with

PDD

made

significantly

less

fixations

than

the

control

group

-LRB-

means

respectively

and

-RRB-

In

addition

there

were

significant

interactions

between

Group

Probe

Group

Display

and

Group

Probe

Display

When

tested

per

level

of

Display

subjects

with

PDD

showed

significantly

fewer

fixations

for

all

display

sizes

-LRB-

F

-LRB-

-RRB-

p

p

p

-RRB-

but

the

effects

were

largest

for

the

set

size

Interactions

of

Group

Probe

effects

were

marginally

significant

for

all

set

sizes

-LRB-

F

-LRB-

-RRB-

p

p

p

-RRB-

indicating

that

group

differences

in

number

of

fixations

were

largest

in

the

target

absent

condition

-LRB-

see

Fig.

-RRB-

Fig.

and

standard

errors

of

the

number

of

fixations

for

each

group

in

trials

with

-LRB-

left

panel

-RRB-

and

without

a

target

-LRB-

right

panel

-RRB-

for

the

easy

and

hard

condition

No

significant

group

differences

were

found

for

fixation

times

at

either

the

first

fixation

or

the

remaining

fixations

Discussion

The

present

study

was

aimed

to

test

two

hypotheses

on

superior

performance

in

visual

search

tasks

in

subjects

with

PDD

It

has

been

suggested

earlier

that

this

superior

performance

reflects

an

enhanced

ability

to

discriminate

between

stimulus

elements

-LRB-

O'Riordan

et

al.

-RRB-

We

tested

this

hypothesis

by

measuring

eye

movements

during

search

tasks

in

high

functioning

adult

men

with

PDD

and

an

age

and

IQ-matched

control

group

In

case

of

enhanced

stimulus

discriminability

shorter

fixation

times

and/or

fewer

fixations

were

expected

in

the

PDD

group

as

compared

to

the

control

group

The

alternative

explanation

that

subjects

with

PDD

use

more

efficient

search

strategies

was

also

tested

A

more

efficient

search

strategy

would

be

reflected

in

a

longer

fixation

time

The

reaction

time

data

showed

that

subjects

with

PDD

were

better

than

the

control

group

for

both

easy

and

hard

search

tasks

especially

in

the

largest

set

size

and

in

trials

in

which

the

target

was

absent

These

findings

are

in

accordance

with

earlier

studies

on

visual

search

in

children

with

autism

indicating

faster

RT

in

the

same

type

of

tasks

-LRB-

O'Riordan

et

al.

Plaisted

et

al.

-RRB-

This

indicates

that

superior

search

performance

in

subjects

with

PDD

is

a

robust

finding

that

can

be

demonstrated

in

both

children

and

adults

Analyses

of

fixations

during

the

search

tasks

showed

that

subjects

with

PDD

made

fewer

fixations

than

controls

This

difference

in

number

of

fixations

was

especially

clear

for

the

largest

set

size

and

for

the

condition

in

which

the

target

was

absent

analogous

to

the

RT

effects

In

addition

it

was

noted

that

many

subjects

with

PDD

showed

an

absence

of

saccadic

eye

movements

in

target

present

trials

indicating

that

they

were

able

to

localize

the

target

in

a

single

glance

No

differences

between

groups

were

found

in

fixation

time

The

absence

of

group

differences

in

fixation

time

indicates

that

subjects

with

PDD

followed

the

same

search

strategy

as

normal

controls

However

the

decreased

number

of

fixations

during

the

task

in

the

PDD

group

is

in

agreement

with

the

hypothesis

of

enhanced

stimulus

discriminability

in

subjects

with

PDD

as

suggested

by

O'Riordan

and

Plaisted

-LRB-

-RRB-

The

finding

that

the

pattern

of

effects

for

fixation

times

was

strongly

similar

to

the

RT

effect

provides

additional

evidence

that

superior

performance

in

this

task

is

indeed

related

to

enhanced

discriminatory

abilities

in

PDD

The

question

what

the

locus

of

this

enhanced

discriminability

could

be

has

been

addressed

by

Cohen

-LRB-

-RRB-

who

tested

a

neural

network

model

of

processing

in

subjects

with

PDD

This

model

showed

that

an

increase

in

processing

units

resulted

in

a

strong

ability

to

discriminate

and

learn

stimulus

patterns

along

with

a

weak

ability

for

generalization

Indeed

there

is

anatomical

evidence

for

abnormal

development

of

the

cyto-architecture

of

the

cerebral

cortex

of

subjects

with

PDD

resulting

in

an

increase

in

processing

units

Recent

post-mortem

studies

in

subjects

with

PDD

show

an

increased

number

of

micro

columns

albeit

of

reduced

size

-LRB-

Casanova

Buxhoeveden

Switala

Roy

Casanova

et

al.

-RRB-

Concluding

the

measurement

of

eye

movements

during

search

tasks

shows

that

the

superior

behavior

of

subjects

with

PDD

in

these

tasks

can

not

be

attributed

to

strategy

difference

but

point

indeed

to

increased

stimulus

discriminability

in

this

group

O O O O O O O O O O O O O O O O B O O B O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B O O O O O O O B O O O O O O O O O O O O O O O B I O O O O O B O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B O O O O O O O O O O O B O O O O O O O O O O O O O O O O O B O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B O O B I O O O O O O O O O O O O O O B O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B O O O O O O O O O O B I O O O O O O O O O O O O O O O O O O O O O O O O O O O O B O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B O O O O O O O O O O O O O O O O O O B I O O O O O O O O O O O O O O O O B O O O O O O O O O O O O O O O O B I O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B O O O O O O O O O O O O O O O O O O O O O O O O O O O O B I O O O O O O O O O O O O O O O B O O O O O O O O O O O O O O O O O O O O O O O O O O O O B O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B O O O O O O O O O O O O O O O B O O O O O O O O O O O O O O O O O O O O O B O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B I O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B O O O O O O O O O O O O O O O O O O O O O O O O O O B O O O O O O O O O O O O O O O O B O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B O O O O O O O O O O O O O O O O O O O O O O O O O B O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B O O O O O O O O O O O O O O O O O O O O O B O O O O O O O O O O O O O O B O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B O O O O O O O O O O O O O O O B O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B O O O O O O O O O O O O O O O O O O O O O O


A steady-state and pre-steady-state kinetics study of the tungstoenzyme formaldehyde ferredoxin oxidoreductase from Pyrococcus furiosus Formaldehyde ferredoxin oxidoreductase from Pyrococcus furiosus is a homotetrameric protein with one tungstodipterin and one -LSB- 4Fe -- 4S -RSB- cubane per 69-kDa subunit . The enzyme kinetics have been studied under steady-state conditions at 80 °C and pre-steady state conditions at 50 °C , in the latter case via monitoring of the relatively weak -LRB- ε ≈ 2 mM − 1 cm − 1 -RRB- optical spectrum of the tungsten cofactor . The steady-state data are consistent with a substrate substituted-enzyme mechanism for three substrates -LRB- formaldehyde plus two ferredoxin molecules -RRB- . The KM value for free formaldehyde -LRB- 21 μM -RRB- with ferredoxin as an electron acceptor is approximately 3 times lower than the value measured when benzyl viologen is used as an acceptor . The KM of ferredoxin -LRB- 14 μM -RRB- is an order of magnitude less than previously reported values . An explanation for this discrepancy may be the fact that high concentrations of substrate are inhibitory and denaturing to the enzyme . Pre-steady-state difference spectra reveal peak shifts and a lack of isosbestic points , an indication that several processes happen in the first seconds of the reaction . Two fast processes -LRB- kobs1 = 4.7 s − 1 , kobs2 = 1.9 s − 1 -RRB- are interpreted as oxidation of the substrate followed by rearrangement of the active site . Alternatively , these processes could be the entry/binding of the substrate followed by its oxidation . The release of the product and the electron shuffling over the tungsten and iron -- sulfur center in the absence of an external electron acceptor are slower -LRB- kobs3 = 6.10 × 10 − 2 s − 1 , kobs4 = 2.18 × 10 − 2 s − 1 -RRB- . On the basis of these results in combination with results from previous electron paramagnetic resonance studies an activation route plus catalytic redox cycle is proposed . Introduction Tungstoenzymes and molybdoenzymes typically catalyze the generic n = 2 reaction : R + H2O ↔ RO + 2 -LSB- H -RSB- , where R is either an oxoanion -LRB- e.g. , sulfite -RRB- or a relatively small organic metabolite -LRB- e.g. , an aldehyde -RRB- . On the basis of structural -LRB- cofactor and protein -RRB- and functional properties molybdoenzymes are classified in three families : the sulfite oxidase family , the xanthine oxidase family , and the dimethyl sulfoxide reductase -LRB- DMSOR -RRB- family -LSB- 1 -RSB- . The latter also encompasses a limited number of tungsten-containing formate dehydrogenases . The majority of the tungstoenzymes , however , can not be classified in any of these three families ; they form a separate family of aldehyde oxidoreductases -LRB- AORs -RRB- together with a few apparently less common AOR molybdoenzyme members -LSB- 2 -RSB- . The mechanism of action of molybdoenzymes has been extensively studied for several decades , especially for xanthine oxidase -LSB- 3 -RSB- . In contrast , attempts to understand the reaction mechanism of tungstoenzymes , in particular for the members of the AOR family , are more recent and of limited extent , although the available structural information on tungstoenzymes and that on molybdoenzymes are comparable . The present study leads to the first proposal of a complete catalytic cycle of the AOR family member formaldehyde oxidoreductase -LRB- FOR -RRB- . The cofactor common to the enzymes of all four families consists of a single Mo -LRB- VI/IV -RRB- or W -LRB- VI/IV -RRB- ion coordinated by the dithioleno sulfurs of one or two three-ring pterin ligands sometimes with an additional nucleotide attached through a phosphoester bond -LSB- 4 -RSB- . All tungsten systems appear to be nonnucleotide dipterin systems with an additional magnesium phosphate coordination between the pterins -LSB- 5 , 6 -RSB- . The coordination number of the Mo or W is 4 -- 7 ; extra ligands are provided by an amino acid side chain and/or by O , S of a small , nonprotein ligand . The vast majority of the Mo/W enzymes have additional prosthetic groups for interaction with a second substrate -LRB- e.g. , NADH -RRB- and/or for electron transfer with a protein natural redox partner -LRB- e.g. , ferredoxin -RRB- . These heme , flavin , and/or iron -- sulfur prosthetic groups are strongly colored to the extent that optical monitoring of the relatively weakly colored -LRB- see below -RRB- metallopterin has thus far not been tried for mechanistic studies of these complex enzymes . Electron paramagnetic resonance -LRB- EPR -RRB- spectroscopy is not a competitive alternative , because the only EPR-detectable oxidation state , Mo -LRB- V -RRB- or W -LRB- V -RRB- , does not appear to be a competent intermediate in the primary , n = 2 reaction . A small group of molybdoenzymes , exemplified by DMSOR , are exceptional because these proteins contain no other prosthetic groups in addition to the active-site molybdopterin . Furthermore , substitution of W for Mo in Rhodobacter capsulatus DMSOR affords an active enzyme with an unmodified 3D structure . The Mo -LRB- VI -RRB- and W -LRB- VI -RRB- metallopterin UV -- vis spectra of this enzyme have been reported to exhibit several broad peaks in the visible and near-UV region with extinction coefficients of the order of ε ≈ 2 mM − 1 cm − 1 -LSB- 7 -RSB- , and the molybdopterin absorption has been used as monitor in a pre-steady-state kinetics study -LSB- 8 -RSB- . Following the DMSOR , the next step up in optical complexity is found in the AOR family of tungstoenzymes , which all contain -- in addition to the tungstopterin active site -- a single -LSB- 4Fe -- 4S -RSB- -LRB- 2 + ; 1 + -RRB- cluster for electron transfer . The visible spectrum of the iron -- sulfur cubane consists of a single broad line at approximately 400 -- 430 nm , which is not only a simpler pattern to that found for -LSB- 2Fe -- 2S -RSB- clusters -LRB- e.g. , in the xanthine oxidase family of molybdoenzymes -RRB- , but it also has a significantly smaller extinction coefficient than flavin or heme prosthetic groups , and so chances are more favorable for monitoring the weaker absorptions from the metallopterin cofactor in these complex enzymes . The homotetrameric -LRB- 4 × 69 kDa -RRB- Pyrococcus furiosus FOR is a member of this family , and we describe here a study of this enzyme as the first example of pre-steady-state kinetics research of a complex , group-6 metalloenzyme using the color of the active center as the monitor . Our choice of FOR was also indicated by the availability of a 3D structure , which was the basis of an initial proposal of part of the enzyme 's working mechanism -LSB- 6 -RSB- , and was further stimulated by the unusual behavior of the enzyme 's substrate in aqueous solution -LSB- 9 , 10 -RSB- . Materials and methods P. furiosus -LRB- DSM 3638 -RRB- was grown at 90 °C under anaerobic conditions with starch as a carbon source , as previously described -LSB- 11 -RSB- . Cells were broken by osmotic shock , diluting with 5 vol 30 mM tris -LRB- hydroxymethyl -RRB- aminomethane -LRB- Tris -RRB- / HCl , pH 8.0 , containing , 0.1 mg ml − 1 DNase I , 0.1 mg ml − 1 RNase , and 1 mM cysteine . A cell-free extract was obtained as the supernatant after 30 min centrifugation at 15,000 g. `` High-activity '' FOR was purified as reported previously -LSB- 9 , 10 -RSB- . Samples were anaerobically purified in 20 mM Tris/HCl buffer , pH 8.0 . An extra column of -LRB- diethylamino -RRB- ethyl -LRB- 2.0 cm × 10 cm -RRB- was equilibrated with 20 mM piperazine/diethanesulfonic acid buffer , pH 6.4 . A 100-ml gradient was used from 0 to 0.5 M NaCl . Ferredoxin was purified as reported previously -LSB- 12 -RSB- . Samples were anaerobically purified in 20 mM Tris/HCl buffer , pH 8.0 . Protein concentration was determined using the bicinchoninic acid method using bovine serum albumin as the standard . Subunit molecular weight and degree of purity were determined with sodium dodecyl sulfate polyacrylamide gel electrophoresis using a Phast system -LRB- GE Healthcare -RRB- . FOR activity was routinely assayed at 80 °C , under anaerobic conditions , with formaldehyde , or glutardialdehyde , as the substrate and 3 mM benzyl viologen as the electron acceptor in 50 mM 4 - -LRB- 2-hydroxyethyl -RRB- -1-piperazinepropanesulfonic acid -LRB- Epps -RRB- buffer , pH 8.4 . Steady-state kinetics of FOR were determined by activity measurements in triplicate in an optical assay with different concentrations of formaldehyde -LRB- 0.5 , 1 , 2 , 5 , 10 , 25 , 50 mM -RRB- as a substrate . Benzyl viologen -LRB- ε600 = 10.6 mM − 1 cm − 1 -LSB- 13 -RSB- -RRB- or ferredoxin -LRB- ε400 = 17.0 mM − 1 cm − 1 -LSB- 14 -RSB- -RRB- were used as an electron acceptor at various concentrations in 50 mM Epps buffer , pH 8.4 , and 80 °C , where one unit is defined as 2 μmol electron acceptor reduced per minute . The concentrations of benzyl viologen and ferredoxin were , respectively , 50 -- 3,000 and 10 -- 200 μM , and the ferredoxin concentration is per dimer -LSB- 15 -RSB- . The UV -- vis spectrum was recorded with a Hewlett-Packard 8452A diode-array spectrophotometer . The optimal excitation wavelength for fluorescence measurements was obtained using a Shimadzu RF-5001PC spectrofluorophotometer . Pre-steady-state studies were carried out under anaerobiosis in 50 mM Epps buffer , pH 8.4 , at 50 °C with formaldehyde -LRB- 25 mM -RRB- and formaldehyde-d2 -LRB- 25 mM -RRB- as a substrate using an SX .18 MV stopped-flow apparatus equipped with a sequential mixing capability and a diode-array rapid-scan detection system -LRB- Applied Photophysics -RRB- . The UV -- vis data were obtained by recording 400 spectra at different time scales -LRB- 16 , 65 and 250 s -RRB- . A spectrum obtained 2 ms after mixing was taken as the `` oxidized '' reference spectrum . Fluorescence data were obtained by exciting the samples at 280 nm , and the change in maximum fluorescence emission was measured at 340 nm . Results Steady-state kinetics Steady-state kinetics studies at 80 °C were performed on the combinations FOR/formaldehyde/benzyl viologen and FOR/formaldehyde/ferredoxin . FOR catalyzes a two-electron transfer from formaldehyde to the one-electron acceptor benzyl viologen or ferredoxin . This results in a reaction with three substrates ; one formaldehyde molecule and two benzyl viologen or two ferredoxin molecules . The possible mechanisms for this reaction are so-called enzyme-substitution mechanisms : the triple-transfer mechanism or the concerted-substitution mechanism -LSB- 16 -RSB- . Triple-transfer mechanism : Concerted-substitution mechanism : The equation for the reaction rate can be simplified , when substrates B and C -LRB- ferredoxin or viologen -RRB- are the same . The results have the same form as equations for the reaction rate of an enzyme with two substrates . The triple-transfer mechanism will transform into the substituted-enzyme mechanism for two substrates and the concerted-substitution mechanism will transform into the ternary-complex mechanism for two substrates -LSB- 16 -RSB- . The apparent KM values for the second and third substrates are identical , and the measured KM is twice the real KM of the second or third substrate -LSB- 16 , 17 -RSB- . To predict whether the systems function according to a ternary-complex mechanism or a substituted-enzyme mechanism , one substrate was varied , while the other substrate concentration was held constant . Substrate inhibition of one of the substrates is described by Eq . 3 , where KsiB is the constant that defines the strength of the inhibition of substrate B with concentration b : Primary plots of the concentration of the inhibitor divided by the reaction rate -LRB- b/v -RRB- versus the concentration of the inhibitor -LRB- b -RRB- are parabolic and intersect at a common point on the b/v axis . The primary plots of the concentration of the noninhibiting substrate divided by the reaction rate -LRB- a/v -RRB- versus the concentration of the noninhibiting substrate -LRB- a -RRB- are linear and intersect at a positive value of the concentration of noninhibiting substrate -LSB- 16 -RSB- . A more complex situation occurs when both substrates show substrate inhibition . At low concentrations of both substrates the shapes of both curves are parabolic . Substrate inhibition by one substrate at low concentrations of the other provides strong positive evidence that the substituted-enzyme mechanism applies -LSB- 16 -RSB- . The plots of a/v versus a and b/v versus b , with substrate A is formaldehyde and B is benzyl viologen or ferredoxin , are presented in Figs. 1 and 2 . All intersection points are situated within experimental error at positive values of the substrates . The parabolic shape of all the figures indicates , as stated above , substrate inhibition by both substrates . All four graphs in Figs. 1 and 2 are consistent with a substrate substituted-enzyme mechanism . Fig. 1Primary plots for Pyrococcus furiosus formaldehyde oxidoreductase with formaldehyde as a substrate and benzyl viologen as an electron acceptor . Formaldehyde was used in concentrations of 1 , 2 , 5 , 10 , 25 , and 50 mM . Benzyl viologen was used at concentrations of 50 , 100 , 500 , 1,000 , 2,000 , and 3,000 μM . Each data point is the average value from three measurementsFig . 2Primary plots for P. furiosus formaldehyde oxidoreductase with formaldehyde as a an substrate and ferredoxin as electron acceptor . Formaldehyde was used at concentrations of 0.5 , 2 , 5 , 10 , 25 , and 50 mM . Ferredoxin was used at concentrations of 10 , 20 , 40 , 80 , 100 , and 200 μM . Each data point is the average value from three measurements One-substrate Michaelis -- Menten kinetics was used to fit the data and to calculate KMapp and Vmaxapp at the different concentrations of substrate and acceptor . To calculate the KM and the Vmax of the different substrates the KMapp and vapp data were fitted using the equations for the apparent values of the Michaelis -- Menten parameters for the substituted-enzyme mechanism -LSB- 16 -RSB- : with equivalent expressions for substrate A . The KM and Vmax values of formaldehyde measured with excess benzyl viologen as an electron acceptor -LRB- Table 1 -RRB- are in agreement with previous measurements -LSB- 10 , 18 -RSB- . The KM value for formaldehyde -LRB- 3.8 mM -RRB- with ferredoxin as an electron acceptor is approximately 3 times less than when benzyl viologen is used as the acceptor . The conversion rate of formaldehyde with ferredoxin as the electron acceptor is 6 times less than when benzyl viologen is used as the acceptor . The values for KM are unusually high for a physiological substrate . We have previously provided experimental evidence indicating that only `` free '' formaldehyde -LRB- i.e. , the nonhydrated form which is 0.2 % at 80 °C -RRB- is a substrate for FOR -LSB- 10 -RSB- . The corrected KM values for free formaldehyde with acceptor benzyl viologen or ferredoxin are , respectively , 71 and 21 μM . Note that Vmax decreases by a factor of 4 when the temperature is lowered from 80 to 20 °C , and that KM for free formaldehyde is temperature-independent -LSB- 10 -RSB- . Table 1 Catalytic properties of formaldehyde oxidoreductase determined at 80 °C with formaldehyde as the substrate and ferredoxin or benzyl viologen as the electron acceptor Electron acceptor Benzyl viologen Ferredoxin KM total formaldehyde -LRB- mM -RRB- 13 ± 2 3.8 ± 0.5 KM free formaldehyde -LRB- μM -RRB- 71 ± 10 21 ± 3 Vmax formaldehyde -LRB- s − 1 -RRB- 28 ± 1a 4.5 ± 0.5 KM electron acceptor -LRB- μM -RRB- 73 ± 19 14 ± 7 Vmax electron acceptor -LRB- s − 1 -RRB- 32 ± 2 6.2 ± 3.2 KM glutardialdehyde -LRB- mM -RRB- 12 ± 2 Vmax glutardialdehyde -LRB- s − 1 -RRB- 13 ± 1 The ratio of activities at 80 °C compared with 50 °C is 2.8 under standard conditions -LRB- see `` Materials and methods '' -RRB- with 50 mM total formaldehyde aIn our previous work -LSB- 10 -RSB- Vmax = 54 s − 1 was reported on a per tungsten atom basis ; this is equal to 28 s − 1 on a per protein molecule basis Varying the formaldehyde concentration gives the KM and Vmax values for the electron acceptors . The KM value of benzyl viologen of 73 μM is comparable to values found in literature for other enzymes that react with benzyl viologen as an electron acceptor . The KM value of ferredoxin -LRB- 14 μM -RRB- is , however , uncharacteristically low : previously reported literature values for FOR and AOR are 100 and 200 μM , respectively -LSB- 9 , 19 -RSB- . An explanation for this discrepancy may be found in the fact that high concentrations of substrate , up to 50 mM of total formaldehyde for FOR , have been used in previous studies to determine KM of ferredoxin . In the present work concentrations from 0 to 10 mM were used to determine KMapp and vapp . When activity was measured with high concentrations of formaldehyde -LRB- i.e. , resulting in significant substrate inhibition -RRB- the apparent KM of ferredoxin increased to approach values found in the literature -LRB- data not shown -RRB- . In a previous study alternative substrates for FOR were reported with KM values of the order of a few millimoles per liter . The lowest KM value was found for glutardialdehyde -LRB- KM = 0.8 mM -RRB- with benzyl viologen as an electron acceptor -LSB- 9 -RSB- . We have been unable to reproduce these results . In our hands the KM for glutardialdehyde was determined to be 12 mM , i.e. , approximately the same as the apparent KM for total formaldehyde . Pre-steady-state kinetics Pre-steady-state kinetics studies were performed on FOR -LRB- 20 μM -RRB- and formaldehyde at 50 °C . Formaldehyde was used at a concentration of 25 mM -LRB- 50 μM free formaldehyde -RRB- . UV -- vis spectra were recorded at 40 - and at 160-ms intervals . At least two spectra per time point were averaged for noise reduction . In the spectrum of oxidized FOR the broad feature of the -LSB- 4Fe -- 4S -RSB- 2 + cluster dominates . However , close inspection reveals weak -LRB- εmax ≈ 1 -- 2 mM − 1 cm − 1 -RRB- absorptions at higher wavelengths similar to those reported for the single-cofactor DMSOR -LSB- 20 -RSB- . After subtraction of the spectrum at t = 2 ms , difference spectra were obtained -LRB- Fig. 3 -RRB- , at 0.1 , 0.5 , 1 , 2 , 5 , 10 , and 16 s , which reveal peak shifts and a lack of isosbestic points . Fig. 3Difference in absorbance versus wavelength measured in pre-steady-state experiments with formaldehyde as a substrate at times 0.1 , 0.5 , 1 , and 2 s -LRB- a -RRB- and 2 , 5 , 10 , and 16 s -LRB- b -RRB- . The reference spectrum was taken at t = 2 ms. c UV -- vis spectra of the oxidized and the substrate-reduced form -LRB- after 65-s incubation time -RRB- of P. furiosus formaldehyde oxidoreductase From the difference spectra five different wavelengths were selected : 335 , 390 , 435 , 595 , and 695 nm . At 595 and 695 nm , approximately corresponding absorption bands can be found in the spectrum of oxidized DMSOR from R. capsulatus -LSB- 8 -RSB- . Amplitude versus time traces of the 40 and 160 ms spectra at the different wavelengths were combined . After 65 s the time traces for all the wavelengths essentially leveled off -LRB- Fig. 4 -RRB- . Data points were taken up to 250 s but no further significant changes were found -LRB- not shown -RRB- . Fig. 4Stopped-flow UV -- vis traces obtained for the reduction of P. furiosus formaldehyde oxidoreductase by formaldehyde at 335 , 390 , 435 , 595 , and 695 nm The major changes at the different wavelengths occur in the first seconds . Three phases starting at different times can be identified in Fig. 5a . The first phase starts at or before t ≈ 20 ms ; the second and the third phases start at approximately 100 and 300 ms , respectively . Changes in the amplitude versus time traces at 390 and 435 nm start at or before 20 ms ; the trace of the wavelength of 595 nm follows the trend of the 335 nm trace , with the difference that all phases start approximately 200 ms later . All data at the different wavelengths were globally simulated with a four-phase exponential least-squares fit -LRB- Table 2 -RRB- . Fig. 5Stopped-flow UV -- vis traces plus fits on a logarithmic time scale obtained for the reduction of P. furiosus formaldehyde oxidoreductase by a formaldehyde or b deuterated formaldehyde at 335 , 390 , 435 , 595 , and 695 nm . c Stopped-flow fluorescence trace plus fit obtained for the reduction of P. furiosus formaldehyde oxidoreductase by formaldehyde at an excitation wavelength of 295 nm and an emission wavelength of 340 nm . d Stopped-flow UV -- vis traces plus fits obtained for the binding of formate by P. furiosus formaldehyde oxidoreductase at 335 , 390 , 435 , 595 , and 695 nmTable 2Rate constants from pre-steady-state kinetics of formaldehyde oxidoreductase plus formaldehyde or deuterated formaldehyde or formate at 50 °C UV -- visFluorescenceCH2OCD2OFormateCH2Ok1 -LRB- s − 1 -RRB- 4.71.120.514.7 k2 -LRB- s − 1 -RRB- 1.90.942.00 × 10 − 21.9 k3 -LRB- s − 1 -RRB- 6.10 × 10 − 26.07 × 10 − 26.10 × 10 − 2k4 -LRB- s − 1 -RRB- 2.18 × 10 − 22.26 × 10 − 2 A previous steady-state kinetics study on the substrate kinetic isotope effect of FOR showed a significant primary isotope effect for C -- H/D bond breaking . The specific activity of FOR dropped by a factor of 3 when deuterated formaldehyde was used as a substrate -LSB- 21 -RSB- . In the present work the pre-steady-state kinetics of FOR with formaldehyde-d2 was determined in the same fashion as for the experiments with formaldehyde . The amplitude versus time traces at selected wavelengths of FOR incubated with formaldehyde-d2 resemble the traces of the different wavelengths when formaldehyde was used as the substrate ; the phases are similar , only the duration and the onset of the changes of states are different . Again the data were fitted with a four-phase exponential fit -LRB- Fig. 5b ; Table 2 -RRB- . Tryptophan residues are excellent probes for protein conformational changes . In FOR a single tryptophan -LRB- Trp441 -RRB- can be found in the active site -LSB- 6 -RSB- . A fluorescence stopped-flow experiment was performed using 280 nm as the excitation wavelength and 340 nm as the emission wavelength . The concentrations of FOR and formaldehyde used were the same as in the previous UV -- vis stopped-flow experiments . Traces were measured on different time scales : 1 , 10 , and 100 s , and were combined -LRB- Fig. 5c -RRB- . The data can be fitted to a three-phase exponential model . The noise levels however are high . The fluorescence data were fitted simultaneously with the UV -- vis data of Fig. 5a , affording a consistent set of parameters -LRB- Table 2 -RRB- . Formaldehyde is oxidized to formate by FOR with reduction of the W -LRB- VI -RRB- center . We checked whether FOR can oxidize formate to carbon dioxide . Activity measurements in an optical assay were done at 80 °C using formate as the substrate and methyl viologen as the electron acceptor . FOR was unable to oxidize formate . Pre-steady-state binding experiments with oxidized FOR using formate as the nonreducible substrate were done at the same time intervals as for the experiments with formaldehyde -LRB- Fig. 5d -RRB- . Although formate is not converted to carbon dioxide by FOR , absorption changes are found in the time traces at the different wavelengths . The first phase starts at t ≈ 20 ms ; the second and the third phases start at approximately 100 and 300 ms , respectively . The time trace at 335 nm resembles the trace obtained with formaldehyde . The time traces at 390 and 435 nm are not similar to each other in contrast to when formaldehyde was used as the substrate . The time traces at 595 and 695 nm are essentially unaffected by the formate . All data at the different wavelengths were globally fitted with a two-phase exponential least-squares model -LRB- Table 2 -RRB- . Discussion Steady-state kinetics An initial proposal for part of the working mechanism of P. furiosus FOR was deduced by Hu et al. -LSB- 6 -RSB- from the enzyme 's 3D structure : two electrons are transferred from the substrate to the tungsten center . The pterin acts as a noninnocent ligand and transfers the electrons via the N8 atom one by one through Cys491 to the iron -- sulfur cluster of FOR . The iron -- sulfur cluster of ferredoxin accepts an electron through its Asp14 -LSB- 6 -RSB- . The electron is then transferred from the iron -- sulfur cluster of ferredoxin to a membrane-bound hydrogenase -LSB- 22 -RSB- . After reduction of the iron -- sulfur cluster of ferredoxin , the electron transfer comes to a halt . Only one electron at a time can pass through this route . Two ferredoxin molecules are necessary to transport both electrons ; alternatively , a ferredoxin dimer -LSB- 15 -RSB- can twice in a row bind to FOR and accept an electron . This means that a two-substrate mechanism is insufficient to describe this three-substrate system . The second and the third substrates are both ferredoxin . This results in steady-state Michaelis -- Menten equations that have the same form as equations for the reaction rate of an enzyme with two substrates ; however , the measured KM for ferredoxin will be twice the `` real '' KM . The triple-transfer mechanism for three substrates with substrates two and three -LRB- ferredoxin -RRB- the same is consistent with the experiments . Both substrates , either formaldehyde and ferredoxin or formaldehyde and benzyl viologen , inhibit FOR . The KM values of 20 -- 70 μM for free formaldehyde are in agreement with our previous studies -LSB- 10 -RSB- . When a nonphysiological electron acceptor such as benzyl viologen is used then the KM for formaldehyde is 3 -- 4 times higher . It is possible that when ferredoxin binds to FOR the enzyme experiences a slight conformational change , resulting in a lower KM for formaldehyde . In these studies a KM of 14 μM was determined for ferredoxin , which is 5 times lower than the KM of benzyl viologen . The high KM reported in the literature for ferredoxin was determined with high concentrations of formaldehyde . An explanation for this high value is that at high concentrations -LRB- 50 mM -RRB- formaldehyde inhibits and partially denatures FOR . Pre-steady-state kinetics Pre-steady-state kinetics studies were performed at 50 °C . This relatively low temperature was dictated by technical limitations of the stopped-flow apparatus . FOR activity at 50 °C is 2.8 times less than at 80 °C , which affords an increased time resolution of enzyme intermediates . Difference spectra from the pre-steady-state data revealed peak shifts and a lack of isosbestic points . These features are an indication that several processes are happening in the first seconds of the reaction . The visible spectrum of the iron -- sulfur cubane consists of a single broad line at approximately 400 -- 430 nm ; the relatively weakly colored metallopterin is found at different wavelengths . A minimal first-order exponential fit model was used to fit the pre-steady-state data . The UV -- vis data could be fitted globally with a three-phase exponential fit . Two fast processes and one slow process were found . The fluorescence data were initially modeled with a two-phase exponential fit , resulting in one fast and one slow process . The fast and slow processes deduced from the fluorescence and from the UV -- vis data were clearly different . A four-phase exponential model afforded a significantly improved fit to the UV-data -LRB- two fast and two slow processes -RRB- , and the fluorescence data were fitted more accurately with a three-phase exponential model -LRB- two fast processes and one slow process -RRB- . The complete set of fluorescence and UV -- vis data obtained with the same enzyme and substrate were then fitted in a global analysis with a three-phase and a four-phase exponential model , respectively . The same four-phase model was used to fit the pre-steady-state data obtained using formaldehyde-d2 as the substrate . The fit is comparable with the fit of formaldehyde but the rate constants of the first and second phases are lower , as expected from the steady-state studies . The first phase is 4 times slower -LRB- k1 = 1.12 s − 1 -RRB- than with formaldehyde as the substrate . The rate constant of the second phase -LRB- k2 = 0.94 s − 1 -RRB- is lower than when formaldehyde is used as the substrate , consistent with a previously reported steady-state kinetics experiment -LSB- 21 -RSB- . The rates for the first two phases with CD2O as the substrate are not significantly different within experimental error . The rate constant for intramolecular electron shuffling appears to be unaffected by the use of formaldehyde-d2 . Residuals of the individual fits plotted versus time were essentially randomly distributed around the x-axis , thus attesting to the quality of the global fit . Several intermediate species are formed during the first seconds of the reaction . These results are in contrast with previous studies on molybdenum -- DMSOR , where in stopped-flow experiments clear isosbestic points were found , and analysis of the time course produced a simple exponential , indicating a simple concerted two-electron reduction of the enzyme with no intermediate species -LSB- 23 -RSB- . Two alternative interpretations can be envisioned for the three phases in pre-steady-state kinetics -LRB- Fig. 6 -RRB- . Fig. 6Proposed alternative models for the pre-steady-state phases of fully oxidized P. furiosus formaldehyde oxidoreductase reacting with formaldehyde . Rate constants with capital letters as a subscript involve binding , oxidizing the substrate , releasing the product , and rearranging electrons between the tungsten center and the iron -- sulfur cluster . Alternatively , rate constants with numbered subscripts involve oxidation of substrate , a conformational change leading to weakened product binding , release of product , and electron rearrangement FOR is represented in Fig. 6 as a rectangle with two redox groups , a tungstopterin -LRB- W -RRB- with oxidation states 6 + , 5 + , or 4 + , and a -LSB- 4Fe -- 4S -RSB- cubane cluster with oxidation states 2 + or 1 + . On the left of W is an empty binding pocket which can be occupied by substrate -LRB- A -RRB- , by product -LRB- P -RRB- , or by ready-to-leave deactivated product -LRB- P * -RRB- . Occupation by A means that the enzyme has acquired a molecule of substrate in the previous reaction . Similarly , a W capped by a double-bonded O means that the enzyme has previously acquired an oxygen -LRB- protonation undefined -RRB- from the solvent . The protein is initially in the fully oxidized form ; the tungsten center is 6 + and has an oxygen atom bound that was previously acquired from a water molecule , and the iron -- sulfur cluster is 2 + . In the first model the initial step -LRB- kA = 4.7 s − 1 -RRB- observed in the optical monitoring is taken to be the overall binding process of formaldehyde to the enzyme . No changes to the formal oxidation state of the tungsten center and iron -- sulfur cluster are assumed to occur in this stage . Subsequently , in what appears to be a single event , the oxygen atom is transferred , the substrate is oxidized to the product -LRB- kB = 1.9 s − 1 -RRB- , and the tungsten center receives two electrons from the substrate and is reduced to 4 + . The product then leaves the protein -LRB- kC = 6.10 × 10 − 2 s − 1 -RRB- . In this step there are no changes in the oxidation states of the tungsten center and the iron -- sulfur cluster . The active site of FOR has been proposed by Hu et al. -LSB- 6 -RSB- to be closed off from the solvent by a lid provided by the protein itself . It is possible that the substrate , free formaldehyde , is so small that it will not experience significant resistance in reaching the active site . The bigger and charged product may have more difficulty getting free of the enzyme , resulting in a low rate . The protein will then , in the absence of an external electron acceptor , redistribute -LRB- to a minor extent ; see below -RRB- its electrons over the tungsten center and the iron -- sulfur cluster -LRB- kD = 2.18 × 10 − 2 s − 1 -RRB- . The tungsten center will become 5 + and the iron -- sulfur cluster 1 + , consistent with previously determined redox properties for `` high-activity '' enzyme -LSB- 10 -RSB- . An isotope effect of 4.2 is found for the rate constant of the first step -LRB- Table 1 -RRB- , which seems to be unrealistically large for a noncovalent binding event , and would rather seem to point toward an event involving the breaking and making of covalent chemical bonds . Therefore , the following would appear to be a rather more likely interpretation of the stopped-flow data . Alternatively , if the entry and binding of the substrate does not lead to a detectable change in the optical absorption , then the first step -LRB- k1 = 4.7 s − 1 -RRB- can be the two-electron reduction of the tungsten . In this model the second step -LRB- k2 = 1.9 s − 1 -RRB- is a conformational change leading to an `` open '' state with decreased affinity for the product formed . The third step -LRB- k3 = 6.10 × 10 − 2 s − 1 -RRB- then is the ready release of this now weakly bound formate . The pre-steady-state phase ends with the two-electron-reduced intermediate which , in the absence of an external electron acceptor , slowly , and only partially converts , by electron shuffling , to the EPR-detectable , paramagnetic side product . Previous EPR studies on FOR revealed this slow electron shuffling at 20 -- 60 °C between the tungsten center and the iron -- sulfur cluster upon reduction with formaldehyde or with dithionite -LSB- 10 -RSB- . Only after minutes the W -LRB- V -RRB- signal appeared in the EPR measurements . This observation is in qualitative agreement with the low rate constants found here for the decay of the fourth state . The pre-steady-state data from the experiments with formate and FOR were fitted with a two-phase exponential model : there is one fast process and one slow process . Formate is not oxidized or reduced by FOR ; therefore , the fast process -LRB- kobs1 = 0.51 s − 1 -RRB- could be the entry and the binding of formate to the active site of FOR . As in the FOR/formaldehyde system the slow process -LRB- kobs2 = 2.00 × 10 − 2 s − 1 -RRB- is probably a nonphysiological process : the protein reaches a dead end . Excluding the very slow electron redistribution step in the absence of external electron acceptor -LRB- the last step in Fig. 6 -RRB- a complete cycle could now be written in which the enzyme shuttles between fully oxidized , one-electron-reduced , and two-electron-reduced , by inclusion of the reoxidation steps of the two-electron-reduced intermediate through serial reactions with two molecules of oxidized ferredoxin . However , such a scheme would be inconsistent with steady-state turnover rates , because the rate constant -LRB- k3 or kC -RRB- of the product-release step -LRB- the penultimate step in Fig. 6 -RRB- is more than an order of magnitude less than kcat . On the other hand , it is conceivable , that the one-electron-reduced intermediate reacts with a second substrate molecule to become three-electron-reduced -LRB- with respect to the resting enzyme -RRB- . This would lead to the cycle presented in Fig. 7 in which the enzyme shuttles between one-electron-reduced , two-electron-reduced , and three-electron-reduced . Fig. 7Proposed model for the catalytic redox cycle of P. furiosus formaldehyde oxidoreductase . A is the substrate formaldehyde ; P is the product formate . The cube-enclosing circle represents the natural redox partner , ferredoxin . Pre-steady-state kinetics measures either the steps with rate constants kA through kD or , alternatively , those with constants k1 through k4 . The fully oxidized enzyme is reductively activated by the reaction with formaldehyde to enter a redox cycle in which the enzyme shuttles between one -- electron-reduced , two -- electron-reduced , and three-electron-reduced states . See the text for additional explanation Under this model the steps observed in the pre-steady-state experiments would actually constitute an activation process . Note that once the steady state of this cycle has been reached , the enzyme reacts sequentially with one formaldehyde and two ferredoxin molecules consistent with the `` A +2 B '' Michaelis -- Menten steady-state analysis . The proposed reaction cycle of Fig. 7 is based on a combination of steady state-kinetics , pre-steady-state kinetics , and EPR studies in the absence of an electron acceptor and available structural data . Future pre-steady-state kinetics studies in the presence of excess natural electron acceptor ferredoxin may put the model to a rigorous , be it experimentally challenging , test .

O O O O O O O O O O O O O B I O O O O B I O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B I O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B I O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B I B I O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B I O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B I O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B I O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B I O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B I O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B I O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B I O O O O O O O O O O O O O O O O O O B I O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B I O O O O O O O O O O O O O O O O O O O O O O O O O O O O B O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B O O O O O O O O O O O O O O O O O O O O O B O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B I O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O

tungsten
formaldehyde oxidoreductase
pyrococcus furiosus

pre-steady-state kinetics
steady-state kinetics