Optical

spectroscopic

studies

of

light-harvesting

by

pigment-reconstituted

peridinin-chlorophyll-proteins

at

cryogenic

temperatures

Low

temperature

steady-state

optical

spectroscopic

methods

were

used

to

study

the

spectral

features

of

peridinin-chlorophyll-protein

-LRB-

PCP

-RRB-

complexes

in

which

recombinant

apoprotein

has

been

refolded

in

the

presence

of

peridinin

and

either

chlorophyll

a

-LRB-

Chl

a

-RRB-

chlorophyll

b

-LRB-

Chl

b

-RRB-

chlorophyll

d

-LRB-

Chl

d

-RRB-

a

-LRB-

a

-RRB-

or

bacteriochlorophyll

a

-LRB-

BChl

a

-RRB-

Absorption

spectra

taken

at

K

provide

better

resolution

of

the

spectroscopic

bands

than

seen

at

room

temperature

and

reveal

specific

pigment

protein

interactions

responsible

for

the

positions

of

the

Qy

bands

of

the

chlorophylls

The

study

reveals

that

the

functional

groups

attached

to

Ring

I

of

the

two

protein-bound

chlorophylls

modulate

the

Qy

and

Soret

transition

energies

Fluorescence

excitation

spectra

were

used

to

compute

energy

transfer

efficiencies

of

the

various

complexes

at

room

temperature

and

these

were

correlated

with

previously

reported

ultrafast

time-resolved

optical

spectroscopic

dynamics

data

The

results

illustrate

the

robust

nature

and

value

of

the

PCP

complex

which

maintains

a

high

efficiency

of

antenna

function

even

in

the

presence

of

non-native

chlorophyll

species

as

an

effective

tool

for

elucidating

the

molecular

details

of

photosynthetic

light-harvesting

Introduction

Photosynthetic

organisms

have

bioengineered

a

wide

variety

of

protein-based

structures

to

carry

out

photosynthetic

light-harvesting

-LRB-

Frigaard

et

al.

-RRB-

High-resolution

molecular

structures

of

many

of

these

systems

are

providing

important

clues

into

the

molecular

features

that

control

light-harvesting

in

photosynthesis

-LRB-

Germeroth

et

al.

Hofmann

et

al.

Hu

et

al.

Koepke

et

al.

Kühlbrandt

et

al.

Law

et

al.

Liu

et

al.

McDermott

et

al.

Roszak

et

al.

-RRB-

To

understand

fully

how

these

naturally

occurring

pigment

protein

complexes

harvest

light

so

efficiently

it

is

necessary

to

employ

spectroscopic

tools

that

reveal

directly

the

dynamics

and

efficiency

of

energy

transfer

and

the

nature

of

the

excited

energy

states

associated

with

the

bound

pigments

The

spectroscopic

studies

are

aided

by

the

ability

to

make

systematic

alterations

in

the

structures

of

the

light-harvesting

complexes

and

then

to

examine

how

these

changes

manifest

themselves

in

the

spectroscopic

observables

dynamics

of

energy

transfer

and

efficiency

of

antenna

function

-LRB-

Akahane

et

al.

Cammarata

et

al.

Chadwick

et

al.

Crielaard

et

al.

Croce

et

al.

Davidson

and

Cogdell

Frank

Fraser

et

al.

Morosinotto

et

al.

Olivera

et

al.

Olsen

et

al.

Plumley

and

Schmidt

Remelli

et

al.

Struck

and

Scheer

Struck

et

al.

-RRB-

The

peridinin-chlorophyll-protein

-LRB-

PCP

-RRB-

complex

from

the

dinoflagellae

Amphidinium

carterae

is

an

example

where

many

of

these

approaches

have

been

applied

Atomic

resolution

structural

analysis

by

X-ray

diffraction

to

Å

resolution

-LRB-

Hofmann

et

al.

-RRB-

has

been

augmented

by

pigment

reconstitutions

and

protein

refolding

studies

-LRB-

Miller

et

al.

Polívka

et

al.

-RRB-

Steady-state

and

time-resolved

spectroscopy

-LRB-

Bautista

et

al.

Kleima

et

al.

Kleima

et

al.

Krueger

et

al.

Linden

et

al.

Shima

et

al.

Zigmantas

et

al.

Zimmermann

et

al.

-RRB-

has

been

carried

out

on

the

excited

singlet

and

triplet

states

associated

with

the

bound

pigments

The

X-ray

crystallographic

analysis

of

PCP

revealed

a

trimeric

structure

of

subunits

-LRB-

Hofmann

et

al.

-RRB-

where

eight

peridinins

and

two

chlorophyll

a

-LRB-

Chl

a

-RRB-

molecules

-LRB-

Fig.

-RRB-

bind

non-covalently

in

a

roughly

symmetrical

arrangement

in

each

of

the

three

individual

subunits

The

peridinins

are

assembled

in

clusters

of

four

within

the

subunits

surrounding

and

in

van

der

Waals

contact

with

one

chlorophyll

a

-LRB-

Chl

a

-RRB-

-LRB-

Fig.

-RRB-

The

two

Chl

molecules

within

a

subunit

have

a

center-to-center

distance

of

Å

Steady-state

and

ultrafast

optical

spectroscopic

investigations

on

peridinin

in

solution

and

on

the

main-form

PCP

-LRB-

MFPCP

-RRB-

have

explored

its

photoinduced

excited-state

processes

and

energy

transfer

pathways

-LRB-

Carbonera

et

al.

Damjanovic

et

al.

Kleima

et

al.

Krueger

et

al.

Ritz

et

al.

Shima

et

al.

Zimmermann

et

al.

-RRB-

In

addition

a

variant

form

the

high-salt

PCP

-LRB-

HSPCP

-RRB-

has

been

studied

in

the

same

manner

-LRB-

Ilagan

et

al.

Sharples

et

al.

-RRB-

Fig.

of

the

pigments

in

a

monomeric

subunit

of

the

MFPCP

complex

The

coordinates

were

taken

from

Protein

Data

Bank

with

code

Also

shown

are

the

structures

of

peridinin

and

the

chlorophylls

used

in

this

study

Adding

to

the

arsenal

of

PCP

complexes

that

can

be

used

for

structural

and

spectroscopic

studies

Miller

and

coworkers

-LRB-

Miller

et

al.

-RRB-

prepared

and

Polívka

and

coworkers

-LRB-

Polívka

et

al.

-RRB-

studied

the

N-domain

and

full-length

PCP

apoproteins

expressed

in

Escherichia

coli

and

reconstituted

with

the

total

pigment

extract

from

native

PCP

to

produce

a

fully

functional

complex

with

a

high

peridinin-to-Chl

a

energy

transfer

efficiency

Also

they

demonstrated

that

in

the

N-domain

PCP

apoprotein

the

bound

Chl

a

molecules

could

be

replaced

by

other

Chls

using

the

N-domain

apoprotein

incorporating

the

requisite

stoichiometric

amount

of

peridinin

In

this

paper

we

present

a

low

temperature

steady-state

optical

spectroscopic

investigation

of

the

spectral

features

of

PCP

complexes

in

which

recombinant

apoprotein

has

been

refolded

in

the

presence

of

peridinin

and

either

chlorophyll

a

-LRB-

Chl

a

-RRB-

chlorophyll

b

-LRB-

Chl

b

-RRB-

chlorophyll

d

-LRB-

Chl

d

-RRB-

a

-LRB-

a

-RRB-

or

bacteriochlorophyll

a

-LRB-

BChl

a

-RRB-

We

use

fluorescence

excitation

spectroscopy

to

examine

in

detail

the

peridinin-to-Chl

energy

transfer

efficiencies

of

the

various

complexes

at

room

temperature

and

correlate

the

results

with

previously

reported

ultrafast

time-resolved

optical

spectroscopic

dynamics

data

-LRB-

Polívka

et

al.

-RRB-

The

low

temperature

data

provide

better

resolution

of

the

spectroscopic

bands

than

seen

at

room

temperature

and

reveal

specific

pigment

protein

interactions

responsible

for

the

observed

Chl

transition

energies

Materials

and

methods

Sample

preparation

The

PCP

complexes

were

reconstituted

from

N-domain

apoprotein

with

peridinin

and

either

Chl

a

Chl

b

Chl

d

a

or

BChl

a

-LRB-

Fig.

-RRB-

as

described

-LRB-

Miller

et

al.

-RRB-

Size

exclusion

chromatography

shows

the

reconstituted

PCP

to

be

approximately

kDa

in

apparent

molecular

mass

i.e.

the

same

as

that

of

the

native

PCP

suggesting

that

the

samples

used

here

consist

of

monomeric

protein

structures

The

purified

reconstituted

PCP

complexes

were

stored

at

°C

until

ready

for

use

in

the

spectroscopic

experiments

For

the

cryogenic

experiments

the

PCP

complexes

were

dissolved

in

a

buffer

containing

mM

Tricine

mM

KCl

buffer

and

-LRB-

v/v

-RRB-

glycerol

at

pH

Chl

a

and

Chl

b

were

obtained

from

Sigma-Aldrich

Chl

d

and

BChl

a

were

extracted

from

the

cyanobacterium

Acaryochloris

marina

and

the

bacterium

Rhodospirillum

rubrum

respectively

Total

pigments

were

extracted

from

A.

marina

cells

with

and

the

extract

dried

in

a

gentle

stream

of

nitrogen

and

Chl

d

was

purified

by

HPLC

as

described

below

BChl

a

was

extracted

from

R.

rubrum

whole

cells

with

-LRB-

v/v

-RRB-

acetone

methanol

The

extracted

solution

was

filtered

dried

and

redissolved

in

-LRB-

v/v

-RRB-

petroleum

ether

methanol

The

methanol

phase

in

which

BChl

a

was

dissolved

was

collected

and

dried

with

nitrogen

gas

The

BChl

a

solution

was

subjected

to

an

alumina

column

chromatography

with

increasing

percent

of

acetone

in

hexane

a

was

obtained

as

an

oxidation

product

of

BChl

a

during

its

isolation

from

R.

rubrum

All

Chls

were

further

purified

using

a

Millipore

Waters

HPLC

system

employing

a

Nova-Pak

column

For

Chl

a

and

Chl

b

isocratic

runs

of

acetone

methanol

water

-LRB-

v/v/v

-RRB-

were

used

as

the

mobile

phase

with

a

flow

rate

of

mL

min

For

Chl

d

and

BChl

a

the

mobile

phase

used

a

combination

of

solvent

A

-LRB-

acetonitrile

methanol

v/v

-RRB-

and

solvent

B

-LRB-

solvent

A

water

v/v

-RRB-

The

run

was

programmed

as

follows

min

linear

gradient

from

solvent

A

and

solvent

B

to

solvent

A

and

solvent

B

and

min

isocratic

solvent

A

and

solvent

B

with

a

flow

rate

of

mL

min

a

was

separated

from

BChl

a

using

solvent

A

-LRB-

acetonitrile

methanol

water

v/v/v

-RRB-

and

solvent

B

-LRB-

hexane

propanol

v/v

-RRB-

as

the

mobile

phase

The

mobile

phase

was

programmed

as

follows

min

linear

gradient

from

solvent

A

to

solvent

A

and

solvent

B

and

min

isocratic

solvent

A

and

solvent

B

with

a

flow

rate

of

mL

min

The

collected

samples

were

dried

with

a

gentle

stream

of

nitrogen

gas

and

then

redissolved

in

tetrahydrofuran

-LRB-

-RRB-

Spectroscopy

Absorption

spectra

were

taken

at

K

using

a

Cary

UV-visible

spectrometer

and

a

Janis

model

helium

vapor

flow

cryostat

using

cm

pathlength

polymethacrylate

cuvettes

The

fluorescence

and

fluorescence

excitation

spectra

of

the

PCP

complexes

were

taken

at

K

using

a

Jobin

Yvon

Horiba

spectrofluorometer

model

on

samples

immersed

in

a

custom

built

optical

dewar

-LRB-

Kontes

-RRB-

as

described

-LRB-

Ilagan

et

al.

-RRB-

Results

Absorption

spectroscopy

The

K

absorption

spectra

of

the

PCP

complexes

reconstituted

with

peridinin

and

either

Chl

a

Chl

b

Chl

d

a

or

BChl

a

are

shown

in

Fig.

All

of

the

complexes

exhibit

strong

absorption

in

the

region

nm

associated

with

peridinin

For

the

complexes

reconstituted

with

Chl

a

and

Chl

b

a

double

peak

is

evident

in

the

Soret

region

-LRB-

near

nm

-RRB-

but

this

is

not

seen

in

complexes

containing

Chl

d

a

or

BChl

a

In

the

long-wavelength

absorption

region

of

the

spectra

the

Qy

band

of

all

the

Chls

is

very

sharp

and

appears

at

nm

for

Chl

b

nm

for

Chl

a

nm

for

a

nm

for

Chl

d

and

nm

for

BChl

a.

All

the

Qy

bands

are

blue-shifted

compared

to

the

spectra

taken

at

room

temperature

Also

except

for

Chl

d

that

exhibits

a

weaker

shift

of

cm

all

are

shifted

by

the

same

amount

cm

Also

in

the

PCP

complex

containing

BChl

a

a

second

peak

on

the

long-wavelength

side

of

the

primary

Qy

feature

is

observed

at

nm

-LRB-

Fig.

-RRB-

and

a

trace

of

a

can

be

seen

in

the

absorption

spectrum

of

the

complex

with

BChl

a.

Fig.

spectra

of

the

PCP

complexes

reconstituted

with

peridinin

and

either

Chl

a

Chl

b

Chl

d

aChl

a

-LRB-

a

-RRB-

or

BChl

a

The

spectra

were

taken

at

K

and

were

normalized

at

nm

For

comparison

and

analysis

of

the

Chl

spectra

of

the

complexes

absorption

spectra

of

the

isolated

and

purified

Chls

in

were

taken

at

K.

forms

a

clear

glass

at

that

temperature

and

has

been

used

previously

in

this

type

of

analysis

-LRB-

Ilagan

et

al.

-RRB-

Figure

shows

the

K

absorption

spectra

of

the

Chl

solutions

in

the

Qy

region

plotted

on

a

wavenumber

scale

The

spectra

were

analyzed

by

Gaussian

deconvolution

and

were

well

fit

using

a

sum

of

two

components

that

were

allowed

to

vary

in

amplitude

position

and

width

built

on

a

broad

baseline

The

Gaussian

deconvolutions

of

the

solution

spectra

were

all

very

similar

-LRB-

Fig.

-RRB-

and

revealed

a

prominent

spectral

origin

and

a

broad

phonon

wing

at

higher

energy

The

wavelengths

of

the

prominent

peaks

computed

from

the

wavenumber

fits

are

nm

for

Chl

a

nm

for

Chl

b

nm

for

Chl

d

nm

for

a

and

nm

for

BChl

a

The

results

indicate

the

chlorophyll

protein

interaction

induces

a

blue-shift

in

the

case

of

Chl

a

Chl

b

and

a

no

shift

for

Chl

d

and

a

red-shift

for

BChl

a.

Fig.

deconvolutions

of

the

absorption

spectra

in

the

Qy

region

of

purified

Chls

in

solvent

The

fit

-LRB-

dashed

line

-RRB-

is

the

sum

of

two

Gaussian

components

built

on

a

broad

background

The

data

for

the

Gaussian

deconvolution

of

the

Chl

a

spectrum

was

taken

from

a

previous

study

-LRB-

Ilagan

et

al.

-RRB-

Figure

shows

the

K

absorption

spectral

profiles

of

the

reconstituted

PCP

complexes

in

the

Qy

region

on

a

wavenumber

scale

fitted

using

Gaussian

functions

that

were

also

allowed

to

vary

in

amplitude

position

and

width

The

spectra

from

the

PCP

complexes

containing

Chl

a

Chl

b

and

BChl

a

required

four

Gaussian

components

on

a

broad

absorption

background

-LRB-

Fig.

B

E

-RRB-

for

a

good

fit

The

spectra

from

the

complexes

having

Chl

d

and

a

required

only

two

Gaussian

functions

on

a

broad

absorption

background

-LRB-

Fig.

D

-RRB-

for

a

good

fit

Fig.

deconvolutions

of

the

absorption

spectra

in

the

Qy

region

of

PCP

complexes

reconstituted

with

peridinin

and

-LRB-

A

-RRB-

Chl

a

-LRB-

B

-RRB-

Chl

b

-LRB-

C

-RRB-

Chl

d

-LRB-

D

-RRB-

aChl

a

-LRB-

a

-RRB-

and

-LRB-

E

-RRB-

BChl

a

Spectral

reconstruction

Linear

combinations

of

the

absorption

spectra

of

peridinin

and

the

various

Chls

taken

in

at

K

were

used

to

reconstruct

the

spectral

line

shapes

of

the

complexes

in

the

region

nm

According

to

previously

published

procedures

-LRB-

Ilagan

et

al.

-RRB-

individual

peridinin

spectra

were

constrained

to

have

identical

intensities

but

were

allowed

to

vary

with

respect

to

wavelength

The

spectra

of

the

individual

Chls

were

allowed

to

have

any

intensity

and

wavelength

For

all

of

the

complexes

two

Chl

and

eight

peridinin

spectra

were

used

to

fit

the

lineshapes

-LRB-

Fig.

E

-RRB-

The

spectra

of

all

the

complexes

were

best

fit

using

two

blue-shifted

peridinins

and

three

separated

pairs

of

peridinin

spectra

-LRB-

Fig.

E

-RRB-

one

of

which

needed

to

be

significantly

red-shifted

to

accommodate

the

long-wavelength

region

of

the

spectra

In

the

complexes

containing

Chl

a

and

Chl

b

the

Soret

bands

of

the

two

Chls

are

split

and

have

noticeably

different

intensities

This

required

two

separate

Chl

spectra

to

reproduce

the

absorption

lineshapes

in

this

region

-LRB-

Fig.

B

Table

-RRB-

No

splitting

of

the

Soret

bands

was

evident

for

the

complexes

with

Chl

d

a

and

BChl

a

so

the

spectra

were

able

to

be

fit

in

this

region

assuming

that

the

two

Chls

were

identical

A

summary

of

the

wavelengths

of

the

spectral

origins

of

the

individual

peridinins

and

the

Chl

bands

assigned

in

this

analysis

is

given

in

Table

Fig.

of

the

absorption

spectra

of

the

reconstituted

PCP

complexes

in

the

region

The

analysis

was

carried

out

by

linearly

combining

individual

K

absorption

spectra

of

peridinin

and

Chl

taken

in

solvent

as

described

in

the

textTable

origins

of

the

eight

peridinins

and

the

positions

of

the

Soret

peaks

and

Qy

bands

of

the

two

Chls

derived

from

the

spectral

analysis

of

the

K

absorption

spectra

of

the

reconstituted

PCP

complexesPCP

-LRB-

Ilagan

et

al.

-RRB-

-LRB-

Ilagan

et

al.

-RRB-

Chl

numbers

are

in

nm

units

and

the

uncertainties

represent

the

range

of

values

that

give

an

acceptable

fit

to

the

dataMFPCP

is

main-form

PCP

HSPCP

is

high-salt

PCP

aChl

a

is

a

is

not

applicable

Fluorescence

spectroscopy

Fluorescence

spectra

of

the

reconstituted

PCP

complexes

at

K

are

shown

in

Fig.

The

spectra

display

a

prominent

narrow

band

at

nm

-LRB-

full

width

at

half

maximum

FWHM

nm

-RRB-

for

Chl

a

nm

-LRB-

FWHM

nm

-RRB-

for

Chl

b

nm

-LRB-

FWHM

nm

-RRB-

for

Chl

d

nm

-LRB-

FWHM

nm

-RRB-

for

a

and

nm

-LRB-

FWHM

nm

-RRB-

for

BChl

a.

All

of

the

spectra

show

structured

vibronic

features

to

the

red

of

the

main

band

In

the

PCP

complex

with

BChl

a

a

shoulder

at

nm

on

the

red

side

of

the

main

peak

is

clearly

evident

Fig.

spectra

of

the

PCP

complexes

reconstituted

with

peridinin

and

either

Chl

a

Chl

b

Chl

d

aChl

a

-LRB-

a

-RRB-

or

BChl

a

taken

at

K

The

spectra

were

normalized

to

their

λmax

values

Fluorescence

excitation

spectroscopy

Figure

shows

the

fluorescence

excitation

spectra

of

the

complexes

recorded

at

room

temperature

overlaid

with

their

spectra

where

T

is

transmittance

The

Chl

fluorescence

was

monitored

at

various

wavelengths

between

and

nm

and

the

fluorescence

excitation

spectra

were

found

to

be

independent

of

the

monitoring

wavelength

The

peridinin-to-Chl

energy

transfer

efficiencies

obtained

from

the

spectra

were

averaged

over

the

nm

wavelength

range

and

were

-LRB-

Chl

a

-RRB-

-LRB-

Chl

b

-RRB-

-LRB-

Chl

d

-RRB-

-LRB-

a

-RRB-

and

-LRB-

BChl

a

-RRB-

Fig.

of

the

room

temperature

fluorescence

excitation

-LRB-

ex

-RRB-

and

-LRB-

where

T

is

transmittance

-RRB-

spectra

of

reconstituted

PCP

complexes

containing

-LRB-

A

-RRB-

Chl

a

-LRB-

B

-RRB-

Chl

b

-LRB-

C

-RRB-

Chl

d

-LRB-

D

-RRB-

aChl

a

-LRB-

a

-RRB-

and

-LRB-

E

-RRB-

BChl

a

The

intensity

of

each

fluorescence

excitation

spectrum

was

normalized

to

correspond

to

the

intensity

of

its

corresponding

spectrum

in

the

Qy

region

Discussion

Low

temperature

absorption

spectra

The

K

absorption

spectra

of

the

reconstituted

PCP

complexes

-LRB-

Fig.

-RRB-

show

significantly

higher

resolution

than

the

spectra

taken

at

room

temperature

-LRB-

Polívka

et

al.

-RRB-

This

allows

a

more

detailed

look

at

how

changes

in

the

structure

of

the

incorporated

Chl

affect

the

spectral

profiles

The

K

lineshape

of

the

PCP

complex

reconstituted

with

Chl

a

-LRB-

Fig.

-RRB-

is

very

similar

to

that

from

the

native

PCP

complex

-LRB-

Ilagan

et

al.

-RRB-

the

major

characteristics

being

the

splitting

of

the

Soret

band

near

nm

and

the

shoulder

on

the

red

side

of

the

peridinin

maximum

These

features

are

clearly

evident

-LRB-

Figs.

-RRB-

in

both

reconstituted

and

native

complexes

-LRB-

Ilagan

et

al.

-RRB-

This

supports

the

notion

that

the

structures

of

these

two

complexes

are

very

similar

The

K

absorption

spectra

of

the

reconstituted

PCP

complexes

-LRB-

Fig.

-RRB-

also

display

blue-shifted

Qy

bands

compared

with

thse

spectra

of

the

complexes

taken

at

room

temperature

and

except

for

Chl

d

that

exhibits

a

shift

of

cm

are

all

shifted

by

cm

This

is

opposite

to

the

low

temperature-induced

red-shift

observed

for

the

nm-absorbing

BChl

molecules

in

complexes

from

photosynthetic

bacteria

which

was

explained

in

terms

of

a

structural

change

of

the

complex

upon

glass

formation

at

low

temperatures

that

increased

excitonic

coupling

between

the

BChls

-LRB-

Wu

et

al.

-RRB-

This

can

not

be

the

case

for

the

PCP

complexes

here

because

the

two

Chl

molecules

are

too

far

apart

-LRB-

Å

-RRB-

to

be

excitonically

coupled

However

the

blue-shift

may

result

from

a

slight

change

in

the

planarity

of

the

Chl

macrocycles

Assuming

that

glass

formation

induces

a

structural

change

-LRB-

Wu

et

al.

-RRB-

in

the

immediate

environment

of

the

Chls

in

the

PCP

complexes

one

may

expect

a

corresponding

change

in

the

conformation

of

the

macrocycles

It

has

been

shown

that

these

conformational

changes

may

induce

either

red

or

blue-shifts

of

Chl

Qy

bands

-LRB-

Gudowska-Nowak

et

al.

-RRB-

The

fact

that

the

shift

is

very

similar

-LRB-

cm

-RRB-

for

all

chlorophyll

species

except

Chl

d

suggests

that

the

structural

change

affects

the

macrocycle

in

the

vicinity

of

Ring

I

because

all

the

Chls

except

Chl

d

have

a

large

substituent

in

the

position

-LRB-

Fig.

-RRB-

Thus

if

one

assumes

a

low-temperature-induced

protein

structural

change

in

the

immediate

environment

of

Ring

I

this

may

lead

to

a

distortion

of

the

Chl

macrocycle

and

consequently

to

the

observed

blue-shift

For

Chl

d

the

smaller

aldehyde

substituent

at

position

may

be

less

susceptible

to

protein

structural

changes

and

to

inducing

conformational

distortion

of

the

Chl

macrocycle

The

Gaussian

deconvolutions

of

the

Chl

solution

spectra

in

the

Qy

region

-LRB-

Fig.

-RRB-

serve

as

guides

in

the

analysis

of

the

absorption

spectra

of

the

reconstituted

PCP

complexes

Comparing

the

spectrum

in

the

Qy

region

of

the

Chl

a-reconstituted

PCP

-LRB-

Fig.

-RRB-

with

that

of

Chl

a

in

solution

-LRB-

Fig.

-RRB-

shows

that

two

distinct

pairs

of

Chl

a

Gaussian

functions

built

on

a

broad

background

are

needed

to

reproduce

the

spectrum

of

that

complex

Likewise

for

the

PCP

complex

containing

Chl

b

two

pairs

of

Gaussian

components

having

different

prominent

spectral

peaks

and

zero-phonon

lines

built

on

a

broad

background

were

needed

to

reproduce

the

spectrum

of

the

protein

complex

-LRB-

Fig.

-RRB-

However

for

the

PCP

complexes

containing

Chl

d

or

a

only

two

Gaussian

components

on

a

broad

background

were

required

to

fit

the

spectra

in

the

Qy

band

region

indicating

that

Chl

d

and

a

in

these

complexes

are

in

highly

symmetric

environments

This

conclusion

is

supported

by

the

observation

that

the

PCP

complexes

reconstituted

with

Chl

a

and

Chl

b

show

a

splitting

in

the

Soret

band

region

-LRB-

Fig.

B

-RRB-

but

all

the

other

complexes

do

not

This

indicates

a

less

symmetric

environment

for

the

Chls

bound

in

the

complexes

containing

Chl

a

or

b

The

observation

that

the

spectra

of

the

PCP

complexes

containing

Chl

a

and

Chl

b

show

splittings

in

the

Soret

and

Qy

regions

attributable

to

environmental

asymmetry

but

the

spectra

from

the

Chls

in

the

other

complexes

do

not

may

also

be

traceable

to

the

different

functional

groups

on

Ring

I

of

the

Chls

A

view

of

the

published

structure

of

the

MFPCP

complex

focusing

on

Chl

in

the

vicinity

of

Ring

I

shows

that

this

part

of

the

Chl

macrocycle

is

in

close

proximity

-LRB-

within

Å

-RRB-

to

peridinins

and

-LRB-

Fig.

-RRB-

The

distance

between

the

vinyl

group

and

the

peridinins

is

closer

than

to

any

amino

acid

residue

in

the

protein

comes

closest

at

Å

-LRB-

Fig.

-RRB-

The

splitting

of

the

Soret

and

Qy

spectral

bands

in

the

Chl

a

and

Chl

b

PCP

complexes

implies

that

the

vinyl

group

on

Chl

a

and

Chl

b

may

adopt

different

configurations

leading

to

distinct

interactions

between

these

Chls

and

the

peridinins

In

contrast

the

carbonyl

groups

on

Ring

I

of

Chl

d

a

and

BChl

a

may

have

only

a

single

configuration

and

may

also

sterically

hinder

the

approach

of

peridinins

and

toward

the

Chl

in

those

complexes

thereby

minimizing

the

interaction

between

pigments

rendering

the

Chl

spectra

equivalent

Fig.

view

of

the

structure

of

the

MFPCP

complex

focusing

on

the

Ring

I

of

Chl

Per

Per

and

Leu

are

in

close

proximity

to

the

Ring

I

of

Chl

The

coordinates

of

the

structure

were

taken

from

Protein

Data

Bank

with

code

The

results

from

the

deconvolution

of

the

spectral

features

for

the

PCP

complex

containing

BChl

a

are

unusual

In

addition

to

the

main

Qy

absorption

band

at

nm

-LRB-

Figs.

-RRB-

a

shoulder

at

nm

is

observed

which

is

highly

reminiscent

of

the

K

absorption

spectrum

of

the

antenna

BChl

a

protein

-LRB-

Fenna

Matthews

Olson

FMO

-RRB-

complex

from

Prosthecochloris

aestuarii

-LRB-

Johnson

and

Small

Wendling

et

al.

Whitten

et

al.

-RRB-

The

spectral

features

in

the

Qy

region

are

attributed

to

a

combination

of

site

shifts

and

dipolar

interactions

among

the

seven

BChl

a

molecules

within

one

subunit

of

the

FMO

protein

-LRB-

Wendling

et

al.

-RRB-

The

crystal

structure

of

this

FMO

complex

has

been

determined

to

Å

resolution

-LRB-

Matthews

et

al.

-RRB-

and

later

refined

to

Å

resolution

-LRB-

Tronrud

et

al.

-RRB-

The

FMO

complex

consists

of

three

identical

subunits

and

each

subunit

contains

seven

BChl

a

molecules

where

the

center-to-center

distances

between

BChls

vary

from

to

Å

-LRB-

Matthews

et

al.

-RRB-

This

is

smaller

than

the

center-to-center

distance

of

Å

reported

from

the

X-ray

crystallographic

analysis

of

the

MFPCP

complex

containing

Chl

a

-LRB-

Hofmann

et

al.

-RRB-

Why

the

BChl-containing

complexes

exhibit

this

long-wavelength

feature

in

their

spectra

but

the

complexes

containing

the

other

Chl

molecules

do

not

is

unclear

Nevertheless

the

Soret

band

and

the

Qy

features

of

the

BChl

molecules

in

the

PCP

complex

are

reproduced

-LRB-

Figs.

-RRB-

assuming

only

a

single

prominent

feature

and

a

phonon

line

Thus

similar

to

the

complexes

containing

Chl

d

or

a

this

suggests

a

highly

symmetric

environment

for

the

BChls

bound

in

this

reconstituted

PCP

complex

The

Chl

protein

interaction

induces

a

blue-shift

of

the

Qy

band

in

the

low

temperature

spectra

of

Chl

a

Chl

b

and

a

no

shift

for

Chl

d

and

a

red-shift

for

BChl

a

compared

to

the

spectra

taken

in

low

temperature

glasses

-LRB-

Figs.

-RRB-

This

may

be

explained

in

a

manner

similar

to

that

used

above

to

interpret

the

blue-shifts

of

the

Qy

bands

induced

upon

lowering

the

temperature

Switching

from

a

glass

to

the

protein

may

induce

a

conformational

change

of

the

macrocycle

that

leads

to

the

observed

shifts

-LRB-

Gudowska-Nowak

et

al.

-RRB-

Structural

differences

between

Chl

species

occur

due

to

different

functional

groups

attached

at

positions

and

-LRB-

Fig.

-RRB-

but

again

it

appears

that

the

site

is

key

The

small

aldehyde

group

of

Chl

d

does

not

provide

sufficient

interaction

leading

to

distortion

of

the

macrocycle

either

in

glass

or

in

the

protein

Thus

essentially

no

protein-induced

shift

is

observed

In

contrast

the

vinyl

group

on

Ring

I

of

Chl

a

and

Chl

b

respond

to

the

protein

environment

and

induce

a

structural

change

that

is

manifested

spectroscopically

as

a

blue-shift

of

the

Qy

band

The

red-shift

of

BChl

a

in

the

protein

can

be

attributed

to

the

acetyl

group

in

the

position

This

functional

group

has

been

shown

to

cause

a

substantial

red-shift

of

absorption

spectra

of

BChl

a

in

complexes

from

photosynthetic

bacteria

-LRB-

Cogdell

et

al.

-RRB-

The

rotation

of

the

carbonyl

group

with

respect

to

the

plane

of

Ring

I

causes

changes

in

π-electron

conjugation

and

this

argument

has

been

used

to

interpret

the

difference

between

the

and

absorbing

BChls

in

different

complexes

-LRB-

Cogdell

et

al.

-RRB-

However

if

this

were

the

sole

effect

a

protein-induced

red-shift

would

be

expected

of

the

a

that

also

has

an

acetyl

group

in

the

site

This

is

not

observed

Thus

we

must

assume

that

the

saturation

of

the

C

C

bond

in

Ring

II

for

BChl

also

plays

a

role

in

determining

the

direction

of

the

shift

Energy

transfer

efficiencies

Light

absorption

by

peridinin

promotes

the

molecule

from

its

ground

state

to

an

excited

state

denoted

The

state

of

peridinin

then

decays

in

fs

to

a

lower-lying

state

which

can

not

be

directly

accessed

from

due

to

symmetry

selection

rules

-LRB-

Akimoto

et

al.

Krueger

et

al.

Zigmantas

et

al.

-RRB-

Since

the

lifetime

of

the

state

of

peridinin

in

solution

was

found

to

be

highly

dependent

on

the

polarity

of

the

solvent

-LRB-

Bautista

et

al.

Zigmantas

et

al.

-RRB-

an

intramolecular

charge

transfer

-LRB-

ICT

-RRB-

state

was

invoked

to

account

for

this

behavior

-LRB-

Bautista

et

al.

-RRB-

and

quantum

computations

have

supported

this

model

-LRB-

Vaswani

et

al.

-RRB-

Moreover

it

has

been

argued

from

pump-dump-probe

experiments

on

peridinin

in

solution

that

and

ICT

states

are

distinct

but

connected

on

a

picosecond

time-scale

-LRB-

Papagiannakis

et

al.

-RRB-

However

other

time-resolved

and

two-photon

spectroscopic

methods

have

suggested

that

it

is

the

itself

that

possesses

the

charge

transfer

character

-LRB-

Shima

et

al.

Zigmantas

et

al.

-RRB-

Since

it

is

not

completely

resolved

whether

the

state

in

PCP

is

distinct

from

or

the

same

as

the

ICT

state

we

shall

use

the

notation

to

indicate

both

possibilities

The

peridinin-to-Chl

energy

transfer

efficiencies

for

the

complexes

can

be

computed

assuming

a

two-state

-LRB-

and

-RRB-

donor

model

-LRB-

Fig.

-RRB-

The

overall

efficiency

ε

is

given

by

Desamero

et

al.

where

and

are

the

rate

constants

for

energy

transfer

from

the

and

states

of

peridinin

is

the

rate

constant

for

internal

conversion

from

to

state

and

is

the

rate

constant

for

to

internal

conversion

The

rate

constants

obtained

directly

from

the

ultrafast

time-resolved

optical

spectroscopic

experiments

carried

out

-LRB-

Polívka

et

al.

-RRB-

are

shown

in

Table

The

value

for

the

rate

constant

for

to

internal

conversion

is

assumed

to

be

ps

-LRB-

fs

-RRB-

from

fluorescence

up-conversion

measurement

on

the

native

PCP

complex

-LRB-

Linden

et

al.

-RRB-

The

value

for

is

assumed

to

be

ps

-LRB-

ps

-RRB-

from

a

measurement

of

the

efficiency

and

dynamics

of

energy

transfer

in

the

native

PCP

complex

-LRB-

Zigmantas

et

al.

-RRB-

The

overall

energy

transfer

efficiencies

calculated

from

Eq

are

compared

with

the

values

obtained

here

from

the

steady-state

measurements

-LRB-

Fig.

-RRB-

and

summarized

in

Table

As

one

can

see

from

the

table

the

efficiencies

determined

using

both

techniques

are

high

in

all

cases

and

also

in

very

reasonable

agreement

with

each

other

Fig.

diagram

of

energy

levels

and

energy

transfer

pathways

between

peridinin

and

Chl

in

the

PCP

complexes

and

-LRB-

solid

lines

-RRB-

are

rate

constants

for

energy

transfer

from

the

and

states

of

peridinin

to

Chl

and

-LRB-

dashed

lines

-RRB-

are

rate

constants

for

internal

conversion

from

to

and

to

respectivelyTable

constants

and

energy

transfer

efficiencies

obtained

from

the

ultrafast

time-resolved

optical

spectroscopic

experiments

on

the

PCP

complexes

reconstituted

with

various

ChlsPCP

-LRB-

ps

-RRB-

-LRB-

ps

-RRB-

-LRB-

ps

-RRB-

-LRB-

ps

-RRB-

ε

-LRB-

from

fluorescence

excitation

-RRB-

ε

-LRB-

from

time-resolved

absorption

-RRB-

Chl

and

are

the

rate

constants

for

energy

transfer

from

the

and

states

of

peridinin

-LRB-

Polívka

et

al

-RRB-

and

are

the

rate

constants

for

internal

conversion

from

to

and

from

to

respectively

was

obtained

from

fluorescence

up-conversion

measurement

on

the

native

PCP

complex

-LRB-

Linden

et

al

-RRB-

was

deduced

from

the

efficiency

and

dynamics

of

energy

transfer

in

the

native

MFPCP

given

in

Ref

-LRB-

Zigmantas

et

al

-RRB-

and

were

assumed

to

be

unchanged

in

all

the

PCP

complexes

The

peridinin-to-Chl

energy

transfer

efficiency

ε

was

obtained

from

steady-state

fluorescence

excitation

experiments

and

from

ultrafast

time-resolved

absorption

spectroscopic

measurements

at

room

temperature

The

uncertainties

in

the

values

of

ε

from

fluorescence

excitation

experiments

were

derived

from

the

standard

deviations

obtained

in

the

nm

wavelength

range

The

uncertainties

in

the

values

of

ε

from

time-resolved

absorption

measurements

were

obtained

by

propagating

the

published

errors

in

the

dynamicsaChl

a

a

In

this

work

it

was

shown

that

optical

spectroscopy

carried

out

at

low

temperatures

provides

a

more

highly

resolved

view

of

the

spectral

features

of

light-harvesting

complexes

than

seen

at

room

temperature

The

steady-state

optical

spectroscopic

methods

applied

to

PCP

complexes

in

which

recombinant

apoprotein

has

been

refolded

in

the

presence

of

peridinin

and

either

Chl

a

Chl

b

Chl

d

a

or

BChl

a

illustrate

the

robust

character

of

the

PCP

protein

which

maintains

high

efficiency

of

peridinin-to-chlorophyll

energy

transfer

function

in

the

presence

of

non-native

Chl

species

The

efficiencies

of

energy

transfer

deduced

from

the

steady-state

measurements

are

in

good

agreement

with

those

from

ultrafast

time-resolved

experiments

The

PCP

complex

continues

to

be

an

effective

tool

for

elucidating

the

molecular

details

of

photosynthetic

light-harvesting

via

systematic

modification

of

its

structural

components

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

absorption
chlorophyll
energy transfer efficiency
fluorescence
low temperature

fluorescence excitation
peridinin-chlorophyll a-protein
reconstitution