J Mol Evol (2004) 59:158–176
DOI: 10.1007/s00239-004-2610-2

Phylogenetic Analysis of Proteins Associated in the Four Major Energy
Metabolism Systems: Photosynthesis, Aerobic Respiration, Denitriﬁcation,and Sulfur Respiration
Takeshi Tomiki, Naruya Saitou
Division of Population Genetics, National Institute of Genetics, and Department of Genetics, School of Life Sciences, Graduate University
for Advanced Studies, Mishima, Japan
Received: 22 September 2003 / Accepted: 28 November 2004

Abstract. The four electron transfer energy metabolism systems, photosynthesis, aerobic respiration,
denitriﬁcation, and sulfur respiration, are thought tobe evolutionarily related because of the similarity ofelectron transfer patterns and the existence of somehomologous proteins. How these systems have
evolved is elusive. We therefore conducted a comprehensive homology search using PSI-BLAST, andphylogenetic analyses were conducted for the threehomologous groups (groups 1–3) based on multiplealignments of domains deﬁned in the Pfam database.
There are ﬁve electron transfer types important for
catalytic reaction in group 1, and many proteins bindmolybdenum. Deletions of two domains led to loss ofthe function of binding molybdenum and ferredoxin,
and these deletions seem to be critical for the electrontransfer pattern changes in group 1. Two types ofelectron transfer were found in group 2, and all its
member proteins bind siroheme and ferredoxin. Insertion of the pyridine nucleotide disulﬁde oxidoreductase domain seemed to be the critical point for theelectron transfer pattern change in this group. The
proteins belonging to group 3 are all ﬂavin enzymes,
and they bind ﬂavin adenine dinucleotide (FAD) or
ﬂavin mononucleotide (FMN). Types of electrontransfer in this group are divergent, but there are two
common characteristics. NAD(P)H works as anelectron donor or acceptor, and FAD or FMN
transfers electrons from/to NAD(P)H. Electrontransfer functions might be added to these common
Correspondence to: Naruya Saitou; email: nsaitou@genes.nig.ac.jp

characteristics by the addition of functional domains
through the evolution of group 3 proteins. Based on
the phylogenetic analyses in this study and previous
studies, we inferred the phylogeny of the energy metabolism systems as follows: photosynthesis (andpossibly aerobic respiration) and the sulfur/nitrogenassimilation system ﬁrst diverged, then the sulfur/nitrogen dissimilation system was produced from thelatter system.
Key words: Energy metabolism — Photosynthesis— Aerobic respiration — Denitriﬁcation — Sulfur
respiration — Protein phylogeny — PSI-BLAST —Pfam — Tree superimposition

Introduction
Photosynthesis, aerobic respiration, denitriﬁcation,and sulfur respiration are energy generation metabolisms. Proton potential is generated through electron transfer, and ATP synthase produces ATP withthe help of this potential in all four systems. Furthermore, the electron transporter in all four metabolism systems is quinone, and cytochrome c also
works as an electron transporter except for sulfur
respiration. Some homologous proteins are used in
diﬀerent metabolic systems. The cytochrome bc1
complex, in aerobic respiration and denitriﬁcation,

159

and the cytochrome b6f complex, in photosynthesis,
are evolutionarily related (Berry et al. 2000; Hauska
et al. 1988). Subunits I and II of cytochrome c oxidase in aerobic respiration were shown to be homologous with nitric oxide reductase subunit b andmonomeric nitrous oxide reductase in denitriﬁcation(Mogi et al. 1998; Saraste and Castresana 1994;
Zumft et al. 1992). Respiratory nitrate reductase,periplasmic nitrogen reductase, and polysulﬁde reductase are homologous between denitriﬁcation andsulfur respiration (Dias et al. 1999; Kraﬀt et al. 1992;
Moreno-Vivian et al. 1999).
It is of interest how these proteins and characteristics common to the four energy metabolism systems
evolved. Evolutionary relationships of some proteinsin the four metabolisms were already analyzed as we
described above. However, an exhaustive phylogenetic analysis of proteins among the four metabolisms has not been done. We therefore conducted amolecular phylogenetic analysis of all the available
amino acid sequences of those four energy metabolism systems using the homology search program
PSI-BLAST (Altschul et al. 1997), because it can
detect evolutionary relationships of highly diverged
proteins (Park et al. 1998). It is possible that homologous proteins in the four energy metabolism
systems were also used in other nitrogen and sulfur
metabolisms (nitrate assimilation, nitrogen ﬁxationand nitriﬁcation in nitrogen metabolism, and assimilation and dissimilation in sulfur metabolism) in
addition to denitriﬁcation and sulfur respiration.
Therefore, we also included amino acid sequences inthese metabolisms for our analysis.
Materials and Methods
Amino Acid Sequence Retrieval
We retrieved amino acid sequences of proteins involved in photosynthesis, aerobic respiration, nitrogen metabolism including
denitriﬁcation, nitrate assimilation, nitrogen ﬁxation, and nitriﬁcation and sulfur metabolism including assimilation and dissimilation from SWISS-PROT (Boeckmann et al. 2003). We referred
mainly to the pathway maps of oxidative phosphorylation, ATPsynthesis, photosynthesis, nitrogen metabolism, and sulfur metabolism in KEGG (http://www.genome.ad.jp/kegg/; Kanehisa et al.
2002). V-type ATP synthase in eukaryotic cells is included in themap of ATP synthesis in KEGG. However, it is not involved in thismetabolism (Wieczorek et al. 1999). Therefore, this enzyme was
excluded from the present study. In total, we used 182 proteins
retrieved by this procedure (see the supplementary information for
the whole list of retrieved proteins).
We used those sequence data as queries and searched SWISSPROT by using PSI-BLAST (Altschul et al. 1997) with SEG ﬁlter
and 10 passes. Based on the PSI-BLAST search result, we deﬁned
homologous groups of query sequences by taking their union. The
E value of 10)3 was used as cutoﬀ to deﬁne homologous proteins.
We found amino acid regions (domains) shared by all the query
sequences in this way. Amino acid sequences retrieved through
PSI-BLAST using these query sequences belonging to one homol-

ogous group are used for phylogenetic analysis. When the functionof a protein was not clearly deﬁned, and was listed only as ‘‘hypothetical protein’’ in SWISSPROT, these sequences were ignoredin further analyses.

Phylogenetic Analysis
Because amino acid sequences which we analyzed are so divergent,
we did not use the whole region of each protein for phylogenetic
analysis so as to obtain reliable multiple alignments and phylogenetic trees. We deﬁned conserved regions based on domains listed
in the Pfam database (http://www.sanger.ac.uk/Software/Pfam/;
Bateman et al. 2002). Pfam is a database of conserved amino acidsequence regions. Each conserved region is called a family. Multiple alignment of conserved region is constructed in each family
and is available from the Pfam Web site. We used multiple alignments available in the Pfam database. When we encountered aminoacid regions which are not deﬁned by Pfam but were weakly homologous with a known Pfam domain, those amino acid sequences
and corresponding Pfam domain sequences were multiply aligned
using ClustalX (Thompson et al. 1997). Neighbor-joining trees(Saitou and Nei 1987) were constructed using MEGA2 (Kumar etal. 2001). Numbers of amino acid substitutions were computed forboth Poisson correction and gamma distance with shape parameter
1 (default value).

Identiﬁcation of Electron Transfer Patterns
In this study, we analyzed the relationship between amino acidsequences and catalytic features mainly focused on patterns ofelectron transfer, because electron transfer is essential for catalysis.
Information on catalytic features, especially about patterns ofelectron transfer, were retrieved from both the SWISS-PROT database and bibliographies cited in this paper. We categorized proteins whose amino acid sequence data were used in this study into
four categories as follows. Categories of amino acid sequence data
are shown in Table 1. References cited in Table 1 are listed insupplementary information.
A. The amino acid sequence of the protein was experimentally
determined, and its function is known. Moreover, electrontransfer pattern in the protein has been revealed or predicted. References regarding its function are shown in
Table 1.
B. The amino acid sequence of the protein was experimentally
determined, and its function is known. References regarding
its function are shown in Table 1.
C. The amino acid sequence of the protein was predicted only
by translating genomic or cDNA nucleotide sequence, andits function was predicted by sequence similarity in SWISSPROT.
D. The amino acid sequence of the protein was predicted only
by translating genomic or cDNA nucleotide sequence, andthere is no description of its function in SWISS-PROT.

Results
Homologous Groups
We found 24 homologous groups of proteins throughthe PSI-BLAST search. Because our objective is to
discover the changes of metabolic pattern among thefour energy metabolism systems and other nitrogenand sulfur metabolisms, we only used homologous

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Table 1. Electron transfer patterns of proteins found to belong to three homologous groups
EnzymeSWISS-PROT ID

Accession no.

Category

Reference(s)aGroup 1
NADH dehydrogenase

Formate dehydrogenase

Nitrate reductase

Polysulﬁde reductase
Thiosulfate reductase
Dimethyl sulfoxide reductase

Trimethylamine–N-oxide
reductase

Biotin sulfoxide reductase

NUOG_ECOLINUOG_RICPR
NUOG_SALTYNUOG_STRCO
NUG2_RHIMENUAM_ACACANUAM_BOVIN
NUAM_HUMAN
NUAM_RECAM
NUAM_SOLTU
NUAM_NEUCR
NQO3_PARDE
NQO3_THETH
FDHA_METFO
FDHA_METJA
FDHF_ECOLI

P33602Q9ZCF6
P33900Q9XAR0
P56914
Q37373P15690
P28331
O21241
Q43644
P24918
P29915
Q56223
P06131
Q60314
P07658

C
C
C
C
C
C
A
B
C
B
B
B
C
B
C
A

FDOG_ECOLIFDNG_ECOLIFDXG_HAEIN
NAPA_DESDE
NAPA_ALCEU
NAPA_ECOLI

P32176
P24183P46448
P81186
P39185
P33937

B
A
C
A
B
B

NAPA_PARPN

Q56350A

NAPA_RHOSHNARB_SYNP7
NARB_SYNY3
NASA_KLEOXNASC_BACSU
NARG_BACSU
NARG_ECOLI

Q53176
P39458
P73448Q06457
P42434
P42175
P09152

B
B
B
B
B
C
A

NARZ_ECOLIPSRA_WOLSUPHSA_SALTYDMSA_ECOLIP19319P31075
P37600P18775B
B
B
A

DMSA_HAEINDMSA_RHOCA

P45004
Q52675CA

DMSA_RHOSH

Q57366A

TORA_ECOLI

P33225A

TORA_SHEMATORZ_ECOLITORZ_HAEIN
BISC_ECOLIBISC_RHOSH

O87948P46923
P44798
P20099P54934B
B
C
B
A

Ohnishi 1998
Chow et al. 1991
Rasmusson et al. 1998
Preis et al. 1991
Xu et al. 1992
Shuber et al. 1986
Boyington et al. 1997;
Sauter et al. 1992
Abaibou et al. 1995
Jormakka et al. 2002
Dias et al. 1999
Siddiqui et al. 1993
Grove et al. 1996;
Thomas et al. 1999
Berks et al. 1995;
Breton et al. 1994;
Butler et al. 1999;
Moreno-Vivian et al. 1999
Reyes et al. 1996
Rubio et al. 1996
Aichi et al. 2001
Lin et al. 1993
Ogawa et al. 1995
Blasco et al. 1989;
Guigliarelli et al. 1992;
Magalon et al. 1997, 1998;
Moreno-Vivian et al. 1999
Blasco et al. 1990
Kraﬀt et al. 1992
Heinzinger et al. 1995
Cammack and Weiner 1990;
Heﬀron et al. 2001;
Rothery et al. 1999;
Rothery and Weiner 1996;
Trieber et al. 1996
Bray et al. 2000;
McAlpine et al. 1998;
Schneider et al. 1996;
Stewart et al. 2000
Li et al. 2000;
Schindelin et al. 1996
Gon et al. 2001
Dos Santos et al. 1998
Gon et al. 2000
Pierson and Campbell 1990
Garton et al. 2000;
Pollock and Barber 2001;
Temple et al. 2000

161
Table 1. Continued
EnzymeSWISS-PROT ID

Accession no.

Category

Reference(s)aCrane et al. 1995, 1997b, cOstrowski et al. 1989b
Bruhl et al. 1996

Group 2
Sulﬁte reductase [NADPH] hemoprotein

Sulﬁte reductase (ferredoxin)

Sulﬁte reductase,assimilatory type
Ferredoxin–nitrite reductase

Nitrite reductase [NAD(P)H]

Anaerobic sulﬁte reductase
Sulﬁte reductase,dissimilatory type

CYSI_ECOLICYSI_SALTYCYSI_THIROSIR_SYNY3
SIR_SYNP7
SIR_DESVH

P17846
P17845
P52673
P72854
P30008Q42590Q05805

A
B
B
C
B
B
A

NIR_SYNP7
NIR_PHOLA
NIR_SPIOLNIR_BETVENIR_NEUCR

P39661
Q51879P05314
P38500
P38681CB
B
CA

NIR_EMENINIRB_KLEOXNASD_BACSUNIRB_ECOLI

P22944
Q06458P42435P08201

B
B
B
B

ASRC_SALTY
DSRA_ARCFU

P26476
Q59109B
B

DSRA_CHRVIDSRB_ARCFU
DSVA_DESVH

O33998Q59110P45574

B
B
B

DSVB_DESVH

P45575B

Gisselmann et al. 1993
Bruhl et al. 1996
Tan and Cowan 1991

Merchan et al. 1995
Back et al. 1988, 1991
Laﬀerty and Garrett 1974;
Prodouz and Garrett 1981;
Vega 1976
Johnstone et al. 1990
Lin et al. 1993
Ogawa et al. 1995
Jayaraman et al. 1987;
Peakman et al. 1990
Huang and Barrett 1991
Dahl et al. 1993
Hipp et al. 1997
Dahl et al. 1993
Karkhoﬀ-Schweizer et al. 1995;
Pierik et al. 1992
Karkhoﬀ-Schweizer et al. 1995;
Pierik et al. 1992

Group 3
CDP-6-deoxy-delta-3,
4-glucoseen reductase
Anaerobic sulﬁte reductase
Benzoate 1,2-dioxygenase
Bifunctional P-450:NADPH-P450
reductase

Sulﬁte reductase [NADPH]
ﬂavoprotein

Phenol hydroxylase
P5 protein
Flavohemoprotein

NADH oxidoreductase hcr
Fruit protein PKIWI502
Flavin reductase
NADH–cytochrome
b5 reductase

Methane monooxygenase

ASCD_YERPE

P37911A

Gassner et al. 1996;
Johnson et al. 1996
Huang and Barrett 1991
Karlsson et al. 2002
Haines et al. 2001;
Hazzard et al. 1997;
Li and Poulos 1997;
Peterson et al. 1997;
Ravichandran et al. 1993;
Sevrioukova et al. 1996, 1999a, b;
Yeom et al. 1995
Eschenbrenner et al. 1995a, b;
Gruez et al. 2000;
Ostrowski et al. 1989a;
Siegel et al. 1973
Hansen et al. 1994
Powlowski and Shingler 1990

ASRB_SALTY
BENC_ACICA
CPXB_BACME

P26475
P07771
P14779B
A
A

CYSJ_ECOLI

P38038A

MT10_YEASTDMPP_PSESP

P39692
P19734B
A

FHP_CANNOFHP_YEAST
HMPA_ALCEU
HCR_ECOLI
K502_ACTCH
LUXG_VIBHA
MCR1_YEAST

Q03331
P39676
P39662
P75824
P43394
P16447P36060

B
B
B
A
D
C
B

Iwaasa et al. 1992
Zhu and Riggs 1992
Cramm et al. 1994
van den Berg et al. 2000

NC5R_BOVIN

P07514B

NC5R_RATP20070B

Ozols et al. 1984, 1985;
Strittmatter et al. 1992
Pietrini et al. 1988;
Zenno et al. 1990

NC5R_YEAST
MMOC_METCA

P38626
P22868C
A

Hahne et al. 1994

Lund et al. 1985

162
Table 1. Continued
EnzymeSWISS-PROT ID

Accession no.

Category

Reference(s)aNADPH–cytochrome
P450 reductase

NCPR_HUMAN

P16435B

Haniu et al. 1989

NCPR_RAT

P00388B

NCPR_YEASTNDOR_PSEPUP16603Q52126

B
B

Porter and Kasper 1985;
Porter et al. 1990
Yabusaki et al. 1988
Simon et al. 1993

NIA_ASPNGNIA_PHYIN
NIA_PICAN
NIA2_ARATH
NOS_DROME
NOS1_RATNOS2_MOUSE

P36858
P39864
P49050
P11035Q27571
P29476
P29477

B
B
B
A
B
B
A

NOS3_BOVIN
NOS3_HUMAN
NS2A_HUMAN

P29473
P29474
P35228B
B
A

PDR_BURCE

P33164

A

PHT2_PSEPUPOBB_PSEPS

Q05182
Q52186B
B

Unkles et al. 1992
Pieterse et al. 1995
Avila et al. 1995
Skipper et al. 2001
Regulski and Tully 1995
Bredt et al. 1991
Aoyagi et al. 2001;
Crane et al. 1997a, 1998, 1999;
Ghosh et al. 1999;
McMillan et al. 2000;
Siddhanta et al. 1998
Lamas et al. 1992
Janssens et al. 1992
Fischmann et al. 1999;
Li et al. 1999
Batie et al. 1991;
Correll et al. 1992
Nomura et al. 1992
Dehmel et al. 1995

RFBI_SALTY
TMOF_PSEMEVANB_PSEPU

P26395Q03304O54037D
B
B

Yen and Karl 1992
Venturi et al. 1998

XYLA_PSEPUXYLZ_PSEPUYEAX_ECOLIFENR_ANASO

P21394
P23101
P76254
P21890A
B
C

FENR_SYNY3

Q55318A

FENR_PEAFENR_SPIOL

P10933
P00455B

FENR_AZOVI

Q44532A

FENR_BUCAPFENR_ECOLI

Q9Z615
P28861C
A

Naphthalene 1,2-dioxygenasesystem, ferredoxin–NAD(+)
reductase component
Nitrate reductase

Nitric-oxide synthase

Phthalate dioxygenase
reductasePhenoxybenzoate
dioxygenase
Toluene-4-monooxygenase
Vanillate O-demethylase
oxidoreductase
Xylene monooxygenase
Toluate 1, 2-dioxygenase
Putative dioxygenase
Ferredoxine–NADP
reductasea

Shaw and Harayama 1992
Harayama et al. 1991
Arakaki et al. 1997;
Martinez-Julvez et al. 1998, 2001;
Mayoral et al. 2000;
Morales et al. 2000;
Serre et al. 1996
Arakaki et al. 1997;
van Thor et al. 1999
Newman and Gray 1988
Jansen et al. 1988;
Karplus et al. 1984
Arakaki et al. 1997;
Isas and Burgess 1994;
Sridhar Prasad et al. 1998
Arakaki et al. 1997;
Bianchi et al. 1993;
Ingelman et al. 1997

References are given in the supplementary information.

groups including proteins belonging to more than one
metabolism system in the further analyses. Homologous groups suitable for our objective were groups 1–
8. Table 2 lists these eight groups. Homologous relationships of groups 5–8 have already been reported(Berry et al. 2000; Hauska et al. 1988; Mogi et al.
1998; Saraste and Castresana 1994). All the proteinsin group 4 had a conserved domain deﬁned as theFer4 domain (4Fe–4S ferredoxin binding domain) by
the Pfam database (Bateman et al. 2002). Unfortunately, this domain is very short (less than 50 aminoacids), and we could not construct a reliable phylogenetic tree. We thus analyze groups 1–3 in the following.

163
Table 2. Eight homologous protein groups found in this study
ID

Name

Metabolism

No. of homologous proteins

O
NDN
NDN
NDN
NDN
SDS
SR

44

NAS
NAS
SDS
SDS
SAS
SAS
SAS

21

Group 1
NUOG_ECOLINARG_ECOLINARB_SYNY3
NASA_KLEOXNAPA_ECOLIPHSA_SALTY
PSRA_WOLSU

NADH dehydrogenase chain G
Respiratory nitrate reductase alpha chain
Assimilatory nitrate reductase (ferredoxin-dependent)
Assimilatory nitrate reductase (NADH-dependent)
Periplasmic nitrate reductase
Thiosulfate reductase PhsA subunit
Polysulﬁde reductase chain A
Group 2

NIR_SYNP7
NIRB_ECOLIDSRA_ARCFUDSRB_ARCFU
ASRC_SALTYCYSI_ECOLISIR_SYNY3

Ferredoxin–nitrite reductase
Nitrite reductase (NAD(P)H) large subunit
Sulﬁte reductase (dissimilatory-type) alpha subunit
Sulﬁte reductase (dissimilatory-type) beta subunit
Anaerobic sulﬁte reductase subunit C
Sulﬁte reductase (NADPH) beta component
Sulﬁte reductase (ferredoxin)

FENR_SYNY3
NIA2_ARATH
NIA_BETVENIA_NEUCRCYSJ_ECOLIASRB_SALTY

Ferredoxin–NADP reductase
Nitrate reductase [NADH]
Nitrate reductase [NAD(P)H]
Nitrate reductase [NADPH]
Sulﬁte reductase (NADPH) alpha component
Anaerobic sulﬁte reductase subunit B

PSAC_SYNY3NUOI_ECOLINARH_ECOLIPSRB_WOLSUPHSB_SALTY
DSRA_ARCFUDSRB_ARCFU
ASRC_SALTY

Photosystem I subunit VII (PsaC)
NADH dehydrogenase chain I
Respiratory nitrate reductase beta chain
Polysulﬁde reductase chain B
Thiosulfate reductase PhsB subunit
Sulﬁte reductase (dissimilatory-type) alpha subunit
Sulﬁte reductase (dissimilatory-type) beta subunit
Anaerobic sulﬁte reductase subunit C

CYB6_SYNY3CYB_YEAST

Cytochrome b6f complex cytochrome b6 subunit
Cytochrome bcl complex cytochrome b subunit

UCRI_SYNY3
UCRI_YEAST

Cytochrome b6f complex iron-sulfur subunit
Cytochrome bc1 complex iron-sulfur subunit

Group 3
P
NAS
NAS
NAS
SAS
SAS

122

Group 4
P
O
NDN
SR
SDS
SDS
SDS
SAS

185

Group 5P
O, NDN

956

P
O, NDN

37

O
NDN

247

P
O, NDN

937

Group 6

Group 7
COX2_YEASTNOSZ_PSEST

Cytochrome c oxidase polypeptide II
Nitrous oxide reductase
Group 8

PETD_SYNY3CYB_YEAST

Cytochrome b6f complex subunit 4
Cytochrome bc1 complex cytochrome b subunit

Note. P, photosynthesis; O, aerobic respiration; NDN, denitriﬁcation; NAS, nitrate assimilation; SR, sulfur respiration; SDS, dissimilation in
sulfur metabolism; SAS, assimilation in sulfur metabolism.

Constellations of the homologous proteins on themetabolic pathway map in the four energy metabolisms (photosynthesis, aerobic respiration, denitriﬁcation, and sulfur respiration) and other metabolisms(nitrate assimilation, dissimilation and assimilation in
sulfur metabolism) are shown in Fig. 1. Black andblue arrows designate electron ﬂow and compoundchange (enzymatic reaction), respectively. Squares areproteins. When one protein is composed by subunits,
small squares showing subunits are shown in thelower part of the square. Only homologous proteins
belonging to more than one metabolism system areshown in Fig. 1. Same-colored proteins are homologous. Because homology search was done by masking
hydrophobic regions, the COX1 Pfam domain (group25 in Fig. 1) was not detected in the present study.

164

Fig. 1. Homologous proteins in the four metabolisms (photosynthesis, aerobic respiration, nitrogen metabolism, and sulfur
metabolism). Contiguous and dotted arrows designate electron
ﬂow and compound change (enzymatic reaction), respectively.
Squares are proteins. When one protein is composed of subunits,
small squares showing subunits are shown in the lower part of thesquare. Some proteins involved in the four metabolisms are notshown in this ﬁgure. Proteins with same symbols are homologous.
The explanatory notes of symbols are given at the bottom. Eachsymbol shows each homologous group. Group 25 proteins (COX1)
were not detected by homology search analysis in this study, butthey are thought to be homologous (Mogi et al. 1998; Saraste andCastresana 1994; Zumft et al. 1992). Subunits A to D and H to N
of NDH are put together. Same alphabetical subunits of NDH inphotosynthesis and NDH in oxidative phosphorylation are homologous. Conserved Pfam domains in each homologous groupare shown in parentheses in the explanatory notes. PS II, photosystem II; NDH, NADH dehydrogenase; PQ, plastoquinone; Cyt
b6f, cytochrome b6f complex; ISP, iron sulfur protein (subunit);
FNR, ferredoxin NADP reductase; Cyt c553, cytochrome c553 ; PS
I, photosystem I; UQ, ubiquinone; Cyt bc1, cytochrome bc1 complex; Cyt c, cytochrome c; COX, cytochrome c oxidase; NAR,
respiratory nitrate reductase; NAP, periplasmic nitrate reductase;
NAS, assimilatory nitrate reductase; Cat, catalytic subunit; Cyt
c550, cytochrome c550 ; NIA, eukaryotic assimilatory nitrate reductase; NIR, nitrite reductase; NOR, nitric oxide reductase;
NosZ, nitrous oxide reductase; MQ, menaquinone; PSR, polysulﬁde reductase; PHS, thiosulfate reductase; DSR, sulﬁte reductase,dissimilatory type; SIR, sulﬁte reductase (ferredoxin); CYS, sulﬁtereductase (NADPH); ASR, anaerobic sulﬁte reductase.

Group 1
Domain compositions of 44 proteins belonging tohomologous group 1 are shown in Fig. 2. All group 1
proteins have the Molybdopterin domain, which hasa binding site to molybdenum (Boyington et al. 1997;

Czjzek et al. 1998; Bias et al. 1999; Jormakka et al.
2002; Li et al. 2000; McAlpine et al. 1998; Schindelin
et al. 1996; Schneider et al. 1996; Stewart et al. 2000).
The phylogenetic tree of this group is presented inFig. 3, based on the multiple alignment of the Molybdopterin domain. Although the nine sequences(FDXG_HAEIN, FDOG_ECOLI, FDNG_ECOLI,
NQO3_THETH, NUG2_RHIME, NUOG_MYCTU, NUOG_ECOLI, NUOG_SALTY, and
NUOG_STRCO) listed in Fig. 2 have the Molybdopterin domain, those domain sequences were
fragmentary. We therefore excluded the nine sequences from the phylogenetic tree (Fig. 3). Topologies of the two NJ trees constructed using Poissoncorrection (not shown) and gamma distance (Fig. 3)
were slightly diﬀerent. We thus constructed a tree
(not shown) using a combination of the Molybdopterin domain and Molydop_binding domain regions.
Although nine proteins lack the Molydop_binding
domain, they formed a clear monophyletic clusters
with a high bootstrap probability (see Fig. 3), andexclusion of those sequences does not inﬂuence theoverall tree topology. This new tree is more reliable
than the tree constructed using only the Molybdopterin domain, with higher bootstrap probabilities(shown in parentheses in Fig. 3). The topology of thisnew tree was consistent with that of the tree computed for the gamma distance, thus we chose that tree
as shown in Fig. 3.
All group 1 proteins are involved in enzymatic
reactions accompanied by electron transfer. Group 1
proteins shown in Fig. 3 can be divided into ﬁve types
based on domain structures corresponding to electron transfer patterns. Arrows in electron transfer
patterns in Fig. 3 designate directions of electron
ﬂow. Electron ﬂow in one pair of brackets occurs ineach group 1 protein.
Type 1 proteins (NADH dehydrogenase subunit)
do not bind molybdenum. Molybdenum plays animportant role for reaction with substrates involving
electron transfer. The main function of type 1 proteins is electron transfer from one subunit to anotherone through three ferredoxins; two 4Fe4Ss and one
2Fe2S (Ohnishi 1998). Only the type 1 protein has
Fer2 domain that has 2Fe2S ferredoxin binding
function.
Type 2 proteins (formate dehydrogenase subunit)
bind molybdenum. Electron ﬂow of this protein is asfollows; electron donor (substrate [formate]) ¼)
molybdenum ¼) 4Fe4S ¼) electron acceptor (another subunit) (Boyington et al. 1997; Jormakka et al.
2002). The Molybdop_binding domain, found formost of the type 2 proteins, is important for binding
molybdenum (Boyington et al. 1997; Czjzek et al.
1998; Dias et al. 1999; Jormakka et al. 2002;
Schindelin et al. 1996; Schneider et al. 1996).
FDHA_METJA lost this domain. The function of

165

Fig. 2. Domain structures of group 1 proteins. SWISS-PROT IDs
were used for protein names. Domains are deﬁned following the
Pfam database. Binding sites of molybdenum (Mo), which is important for catalysis, are shown by ﬁlled circles. We predicted

molybdenum binding sites based on the multiple alignment ofgroup 1 proteins. We regarded amino acids in the same column as
known molybdenum bindings site as the molybdenum binding sites(shown by shaded circles).

FDHA_METJA was predicted only by sequencesimilarity (Table 2). FDHA_METJA may not bindmolybdenum. Type 3 proteins (subunit of assimilatory and periplasmic nitrate reductase) have the same
domains as type 2 proteins except for
FDHA_METJA, but the electron ﬂow is reversed(Breton et al. 1994; Butler et al. 1999; Dias et al.
1999). 4Fe4S ferredoxin is bound to the Molybdop_Fe4S4 domain, the 4Fe4S ferredoxin binding
domain, in proteins of types 2 and 3.
Type 4 proteins (subunit of the trimer-type DMSO
reductase and respiratory nitrate reductase) do notbind 4Fe4S ferredoxin. The Molybdop_4Fe4S domain of the trimer-type DMSO reductase subunit(DMSA_ECOLI and DMSA_HAEIN) is less conserved, and that of the respiratory nitrate reductase
subunit (NARG_BACSU, NARG_ECOLI and
NARZ_ECOLI) was lost. Four cysteines in this domain are essential for binding 4Fe4S ferredoxin. The
arrangement of the four cysteines in type 4 is diﬀerent
from that of types 2 and 3 (Trieber et al. 1996). There
are two amino acids between the ﬁrst N-terminus two
cysteines in type 2 and 3 proteins, but there are threeamino acids in the trimer-type DMSO reductase
subunit. In the respiratory nitrate reductase subunit,

the ﬁrst cysteine is replaced by histidine. Changed
cysteine arrangement in type 4 lost the ability to bindferredoxin (Magalon et al. 1998; Trieber et al. 1996).
The cysteine arrangement is not found in type 5
proteins (biotin sulfoxide reductase, trimethylamineN-oxide reductase, and monomer-type DMSO reductase), thus type 5 does not bind ferredoxin.
Although electron transfer patterns of types 4 and5 look the same (see Fig. 3), electron transfer patterns
among electron donor, molybdenum, and electron
acceptor seem to be diﬀerent between these two types.
A type 4 protein gets an electron from the electron
donor (substrate) at active site and probably gives itto the electron acceptor (another protein) by way of
another side (Rothery et al. 1999). In contrast, a type5 protein gives an electron to the electron acceptor at
the same active site (Schindelin et al. 1996).
Which type of electron transfer pattern was ancestral among the ﬁve types in group 1 proteins? Type2 proteins exist in eubacteria and archaea. This suggests that type 2 proteins emerged before the divergence of eubacteria and archaea. Types 3–5 proteinsare found only in eubacteria (see Fig. 3), suggesting
their relatively recent origin, after the divergence of
eubacteria and archaea. Type 1 proteins exist in both

166

Fig. 3. A phylogenetic tree of group 1 proteinsbased on Molybdopterin-domain sequences. The
shaded region shows possible locations of roots
for this tree. Each square designates a Pfam
domain. Explanatory notes on square symbols
are given in Fig. 1. Electron transfer patterns arealso shown. Arrows of the patterns indicate thedirection of electron ﬂow. D and A in squares:
mean electron donor and acceptor, respectively.
Unknown patterns of electron transfer werepredicted from known patterns of evolutionarilyclosely related proteins. Proteins with an asterisk
can generate a proton gradient.

eubacteria and eukaryotes. Type 1 protein genes insome eukaryote species (NUAM_ACACA and
NUAM_RECAM) are encoded in their mitochondrial genomes. It is hypothesized that mitochondria
was established as a result of endosymbiosis of anancestor of the alpha proteobacterium P. denitriﬁcans, and it is also hypothesized that the type 1 protein gene encoded in the mitochondrial genome wastransferred to the eukaryotic genome after theendosymbiosis (Finel 1998). Andersson et al. (1998)
showed that the alpha proteobacterium R. prowazekii
is the closest eubacterial relative of mitochondria.
Therefore, the type 1 protein gene of eukaryotes
could be derived from that of the ancestor of alphaproteobacteria. Consequently, type 2 proteins seemto be the ancestor type protein in group 1. The graycolored region in Fig. 3 shows the possible locationof the root for this tree. We could not determine theexact position of the root.
Group 2The domain structures of 21 proteins belonging togroup 2 are shown in Fig. 4. All the group 2 proteins

have the NIR_SIR Pfam domain and the NIR_SIR_ferr Pfam domain in adjacent locations. Bothdomains have siroheme binding sites. It should be
noted that, confusingly, the NIR_SIR domain, not
the NIR_SIR_ferr domain, have ferredoxin bindingsites according to Pfam annotation (Crane et al.
1995). Hatched domains in Fig. 4 are not deﬁned by
Pfam, but they were detected as weakly similar tothese two Pfam domains through PSI-BLAST search.
Some of them were previously noticed by Crane andGetzoﬀ (1996) and Larsen et al. (1999). We constructed a phylogenetic tree of group 2 proteins using
the conserved regions consisting of the two domains(Fig. 5). The ﬁrst nine proteins in Fig. 4 have two
NIR_SIR_ferr and two NIR_SIR domains, althoughferredoxin binding sites are lost in hatched NIR_SIR
domains (Crane et al. 1995). These two NIR_SIR_ferr and two NIR_SIR domains suggest that an internal gene duplication occurred. We thus used eachregion as independent sequence in the case of these
duplicated conserved regions. For example, we used
two sequences in diﬀerent conserved regions of
CYSI_ECOLI; amino acid positions 109 to 233 and387 to 489.

167

Fig. 4. Domain structures of group 2 proteins. SWISS-PROT IDs were used for protein names. Domains are deﬁned following the Pfam
database. Domains F and H were found in this study.

Fig. 5. A phylogenetic tree of group 2
proteins based on conserved regions
consisting of the NIR_SIR_ferr domain and
NIR_SIR domain. Conserved regions which
are used for constructing this phylogenetic
tree were obtained from previous works(Crane et al. 1997; Crane and Getzoﬀ 1996;
Larsen et al. 1999). Amino acid sequences of
sulﬁte reductase (Q42590) from previous
works are also added. The shaded region
shows possible locations of roots for this tree.
Each square in this ﬁgure shows a domain.
Explanatory notes for each domain are given
in Fig. 3. Arrows of electron transfer patterns
indicate the direction of electron ﬂow. D and
A in squares: mean electron donor andacceptor, respectively. Unknown patterns ofelectron transfer were predicted from known
patterns of evolutionarily closely related
proteins.

Enzymatic reactions of group 2 proteins are accompanied by electron transfer. Arrows show electron ﬂows. Group 2 proteins can be categorized into
two types by the diﬀerences in electron transfer pat-terns (Fig. 5). Type 1 proteins bind siroheme and
4Fe4S ferredoxin, and electrons from the donor aretransferred to the acceptor at these molecules (Craneet al. 1997; Tan and Cowan 1991). Type 2 proteins

168

Fig. 6. Domain structures of group 3 proteins. SWISS-PROT IDs were used for protein names. Domains are deﬁned following the Pfam
database.

bind siroheme and 4Fe4S ferredoxin. However, electron ﬂow is probably diﬀerent from type 1 as follows;electron donor ¼) FAD ¼) 4Fe4S ¼) siroheme ¼)
electron acceptor or substrate (Vega 1976). The tree
topology of the phylogenetic tree shown in Fig. 5
suggests that the electron transfer pattern for type 1 isancestral and that for type 2 is derived.
Larsen et al. (1999) noticed that the dissimilatory
sulﬁte reductase alpha subunit (DSAR_ARCFU) andbeta subunit (DSRB_ARCFU) of Archaeoglobus
fulgidus may have been duplicated from an ancestraldsr gene before the divergence of eubacteria andarchaea. The tree topology in Fig. 5 indicates that
this gene duplication was followed by the emergenceof proteins having a pair of NIR_SIR and NIR_SIR_ferr domains. This suggests that duplicatedgenes were fused in the ﬁrst nine proteins in Fig. 4.
The root of the tree shown in Fig. 5 must be placed
before this gene duplication. The gray-colored regionin Fig. 5 thus shows the possible location of root forthis tree.
Group 3
Homologous group 3 contains 122 proteins, andthey were found using seven query sequences. The

conserved region shared by all the proteins in thisgroup was the NAD_binding_1 Pfam domain. The
NAD_binding_1 domain has NAD(P)H connection
sites. NAD(P)H works as electron donor or acceptorof the group 3 proteins. Because some proteins wereclosely related and formed clear monophyletic clusters in the phylogenetic tree when we used all proteins, we chose one amino acid sequence from eachcluster. When more than one enzyme was found in
one cluster, we chose representative proteins fromeach enzyme. A total of 48 proteins was thus used in
the following analyses.
Domain structures of group 3 proteins are shownin Fig. 6, and they are all ﬂavin enzymes which bindﬂavin adenine dinucleotide (FAD) or ﬂavin mononucleotide (FMN). FAD is bound to the
FAD_binding_6 domain or the FAD_binding_1 domain, and FMN is bound to the Flavodoxin or the
FAD_binding_6 domain.
Enzymatic reactions of group 3 proteins are accompanied by electron transfer. Arrows in Fig. 7
are electron ﬂows. Group 3 proteins can be categorized into 10 types based on electron transfer
patterns (Fig. 7). Type 1 proteins (ferredoxin–
NADP reductase) have a relatively simple domain
structure, and both eukaryotes and eubacteria have

169

Fig. 7. A phylogenetic tree of group 3 proteins
constructed based on NAD_binding_1 domain
sequences. Each square shows a domain.
Explanatory notes for each domain are given in
Fig. 5. Arrows of electron transfer patterns
indicate the direction of electron ﬂow. D and A
in squares mean electron donor and acceptor,
respectively. Unknown patterns of electrontransfer were predicted from known patterns ofevolutionary closely related proteins.

this type. Furthermore, type 1 proteins are notmonophyletic according to the tree topology(Fig. 7). Therefore, the common ancestor of group 3
proteins could have the type 1 electron transfer
pattern.
Type 2 proteins (nitric oxide reductase) are homodimers. FAD, FMN, and heme are bound to each
subunit. One subunit gets electron from NADPH andgives it to heme of the other subunit through FAD
and FMN of the ﬁrst subunit. Heme of the second
subunit gives electron to electron acceptor. Electronfrom NADPH passes through two subunits and goesto electron acceptor. Electron ﬂow in each pair ofbrackets shows electron ﬂow occurring in each subunit.
Although type 3 proteins bind FAD, FMN, andheme as in the case of type 2 proteins, their electrontransfer pattern is diﬀerent from that of type 2 proteins. Type 3 proteins, are not homodimers. They
receive an electron from NADPH and give it to theelectron acceptor through FAD, FMN, and heme.

The NO_synthase domain of type 2 proteins and theP450 domain of type 3 proteins have heme bindingsites. This suggests that the insertion of theNO_synthase domain or the P450 domain enabledgroup 3 proteins to bind heme. Types 2, 3, and 4
proteins bind FAD and FMN. The FAD_binding_1
domain has FAD binding sites, and the Flavodoxin
domain has FMN binding sites. Insertion of the
Flavodoxin domain may have enabled group 3 proteins to bind FMN.
Figure 8A shows the phylogenetic tree of theproteins belonging to types 2–5, constructed based on
the FAD_binding_6 and NAD_binding_1 domains.
The topology of this tree is more reliable than that of
the tree constructed based on only NAD_binding_1
domain sequences. Type 5 proteins do not have the
Flavodoxin domain. Therefore, the tree in Fig. 8A
suggests that insertion of the Flavodoxin domain
occurred on the lineage going to types 2, 3, and 4
proteins. Figure 8A also suggests that NO_synthase
and P450 domains that have binding sites of heme

170

Fig. 8. A A phylogenetic tree of the type 2, 3,
4, and 5 proteins based on the FAD_binding_1
and NAD_binding_1 domains. B A
phylogenetic tree of the type 1 proteins in group
1 and the type 6 and 8 proteins in group 3 basedon the Fer2 domain. C A phylogenetic tree ofthe type 7 proteins constructed based on the
Globin domain. D A phylogenetic tree of thetype 9 and 10 proteins based on the
FAD_binding_6 and NAD_binding_1 domains.

were inserted on the lineage to type 2 and type 3
proteins, respectively.
Types 6 and 8 proteins both bind 2Fe2S ferredoxin. Ferredoxin is bound to the Fer2 domain.
This suggests that insertion of the Fer2 domain
enabled group 3 proteins to bind ferredoxin. Figure 8B shows the phylogenetic tree constructedbased on the Fer2 domain amino acid sequences.
Not only types 6 and 8 of group 3 but also type 1
proteins of group 1 have this domain. Proteins of
this tree can be largely categorized into two lineages; type 1 proteins in group 1 and types 6 and 8
proteins in group 3. The tree topology of group 3
proteins in Fig. 8B suggests that the Fer2 domain
was ﬁrst inserted to form type 6 proteins and wasfurther inserted to form type 8 proteins. The inserted location of the Fer2 domain is diﬀerent between type 6 and type 8 proteins except for
HCR_ECOLI (Figs. 5 and 8B). Although the domain structure of HCR_ECOLI is similar to that oftype 8 proteins, the electron transfer pattern of
HCR_ECOLI is similar to that of type 6 proteins.
HCR_ECOLI is phylogenetically closer to type 6
proteins (see Fig. 8B). One possibility is that domain inversion occurred in the HCR_ECOLI lineage.
Hemes in type 7 proteins are bound to the Globin
domain (see Fig. 7). The insertion of the Globin domain may have enabled group 3 proteins to bindheme. Type 7 proteins are not monophyletic;

171

Fig. 9. Estimated domain
insertion/deletion events
during evolution of thethree protein groups. Eachsquare shows a domain
deﬁned by Pfam. A Group 1
proteins. Square symbols
are the same as in Fig. 1.
B Group 2 proteins. The
correspondence between
square symbols and Pfam
domain is shown in Fig. 3.
C Group 3 proteins. Squaresymbol of each domain is
the same as in Fig. 5.

172

Fig. 10. Phylogenetic trees of proteins shown in Fig. 9 (upper
trees). Branch length has no meaning. The dashed lines indicate
divergence between eubacteria and others (archaea and eukaryotes). Divergences between eubacteria and others were estimated inthis study (Figs. 2, 4, and 6) and previous studies (Berry et al. 2000;
Saraste and Castresana 1994). The lower trees are superimposed

trees of the upper trees. Each OTU name is the metabolism inwhich each protein works: P, photosynthesis; O, aerobic respiration; NDN, denitriﬁcation; NAS, nitrate assimilation; SR = sulfur
respiration; SDS, dissimilation in sulfur metabolism; SAS, assimilation in sulfur metabolism.

HMPA_ALCEU (bacteria) and FHP_YEAST(eukaryote) formed a weak monophyletic cluster(the bootstrap probability is only 30%), while
FHP_CANNO is located in a diﬀerent lineage (seeFig. 7). Figure 8C shows the phylogenetic tree of type7 proteins constructed based on the Globin-domainamino acid sequences. This tree also suggests that
HMPA_ALCEU and FHP_YEAST form a monophyletic cluster. The bootstrap probability of thiscluster is now 93%. Therefore, it is possible that thelineage of HMPA_ALCEU and FHP_YEAST is
separated from that of FHP_YEAST. This implies
that the Globin domain may have been inserted independently on the lineage to HMPA_ALCEU and
FHP_YEAST and the lineage to FHP_YEAST.
Type 9 proteins are nitrate reductase, and theybind FAD according to the tertiary structure analysis(Lu et al. 1994; 1995). It was hypothesized by sequence similarity that heme and molybdenum arebound to the Heme_1 domain and Oxidored_molyb
domain of type 9 protein, respectively (Avila et al.
1995; Crawford et al. 1988; Pieterse et al. 1995; Unkles et al. 1992). This suggests that insertion of the
Oxidored_molyb domain enabled group 3 proteins tobind molybdenum, and insertion of the Heme_1 domain enabled group 3 proteins to bind heme. Figure 8D shows the phylogenetic tree of type 9 and type

10 proteins constructed based on the NAD_binding_l
and FAD_binding_6 domains. The topology of thistree is more reliable than that of the tree in Fig. 7,
which was constructed based on only NAD_binding_1 domain sequences. This tree suggests that type
9 and 10 proteins are not monophyletic. However,bootstrap probabilities of various branches are nothigh, so it is not clear.
Discussion
Domain Change and Electron Transfer Pattern
Change Through Evolution
The phylogenetic analysis of the three groups suggests that each homologous group experienced frequent insertions and deletions of domains. We thus
estimated domain insertion/deletion events during
evolution and connected those events with electrontransfer pattern changes.
Group 1
We hypothesized the evolutionary process of group
1 proteins, as depicted in Fig. 9A, based on the phylogenetic tree shown in Fig. 3. We assumed that type 2
was the ancestral type of electron transfer pattern, and173

the domain structure of the ancestral protein was assumed to be the same as that of type 2 proteins. Type 2
proteins have Molybdop_Fe4S4 and Molybdopterin
domains. Two of the type 2 proteins have the Molydop_binding domain, which is important for binding
molybdenum, but FDHA_METJA does not have thisdomain. As we described above, we suspected that
FDHA_METJA does not bind molybdenum and lostthe catalytic activity. Because some NADH dehydrogenase subunits (type 1 proteins) have the Molydop_biding domain (Fig. 2), it seems that the ancestralprotein also had this domain. Therefore, we assumed
that the Molydop_binding domain was lost independently in the lineage to FDHA_METJA and on thetype 1 proteins (Fig. 9A). If this hypothesis is true, theancestral protein probably had the Molydop_Fe4S4,
Molybdopterin, and Molydop_binding domains
as is the case for the type 2 proteins except for
FDHA_METJA. The ancestral protein may havebound molybdenum and 4Fe4S ferredoxin.
A scenario of evolutionary changes of the group 1
proteins from this ancestral protein is as follows. On
the lineage to type 1 proteins (NADH dehydrogenase), the Fer2 domain was acquired, and ferredoxin
was bound to this domain. In contrast, the Molybdopterin domain became less conserved, and mosttype 1 proteins lost the Molydop_binding domain.
Both the Fer2 and the ferredoxin domains are important for molybdenum binding. Therefore type 1
proteins lost the function of binding molybdenum,
which is important for catalysis. Their main functionchanged from catalysis to electron transfer. On thelineage to type 3, the direction of electron ﬂow wasreversed. This reversal did not seem to be caused bydomain composition changes. Instead, amino acidsubstitutions for changing reactant speciﬁcities probably occurred on the lineage to type 3, and thechanging of reactants caused the reversal of electron
ﬂow direction. The molybdop_4Fe4S domain that hasbinding sites of 4Fe4S ferredoxin became less conserved on the lineage to type 4 proteins and wascompletely lost on the lineage to type 5. Types 4 and 5proteins lost the function of binding 4Fe4S ferredoxin. In type 5 proteins, electron is exchanged at molybdenum from electron donor to electron acceptor.
There are no other electron carriers in this protein.
The common feature of the group 1 proteins isthat all of them have the Molybdopterin domain(Fig. 2), although this domain is not conserved wellin type 1 proteins. Other features are not necessarily common. For example, ferredoxin is attachedto types 1–3 proteins but not to others (Fig. 3).
Molybdenum is attached to types 2–5 proteins but
not to type 1 (Fig. 3). Some group 1 proteins (indicated by asterisks in Fig. 3) are subunits of proteins that can generate the proton gradient. The
proton gradient is the energy source for generating

ATP by ATP synthase. Based on our phylogenetic
analysis, this important function, proton gradient
generation, is not necessarily conserved. Although
the general electron transfer patterns are more orless similar in type 3 proteins, some proteins(polysulﬁde reductase subunit and thiosulfate reductase subunit) work for proton gradient generation, but the others (assimilatory nitrate reductase
subunit) do not (see Fig. 3).
Group 2
We conjectured the evolution of group 2 proteinsas shown in Fig. 9B. The common ancestral protein
was assumed to have both the NIR_SIR_ferr and the
NIR_SIR domains, because all group 2 proteins havethese two domains including weakly conserved ones.
The two Fer4 domains were assumed to exist in thecommon ancestral protein, as in the case of the
ASRC_SALTY protein. Under this scenario, threelosses of the Fer4 domain were assumed (Fig. 9B).
However, proteins with only one or no Fer4 domain
could also be the common ancestor. All three possibilities are equally parsimonious for the three insertion/deletion events of the Fer4 domain.
Later, a gene duplication produced two copies of
the NIR_SIR_ferr and NIR_SIR domains. Thismeans that one gene including the NIR_SIR_ferr and
NIR_SIR domains was duplicated, and two genes
like a pair of DSRA (alpha subunit of sulﬁte reductase) and DSRB (beta subunit), or DSVA (alphasubunit of sulﬁte reductase) and DSVB (beta subunit), were produced. These two genes were later
fused. In the lineage of DSRA and DSVA, thePyr_redox domain was added and the FAD bindingsite was generated. This creation of the FAD binding
capacity seems to be the critical point for generatingtype 2 proteins.
Group 3A parsimoniously hypothesized evolutionary history of group 3 proteins is shown in Fig. 9C. As we
described in the Results, the electron transfer patternof the ancestral proteins seems to have been type 1. If
so, that ancestral protein had NAD_binding_1 and
FAD_binding_6 domains like type 1 proteins of eubacteria.
On the lineage to types 2–4, the Flavodoxin
domain having the FMN binding region was inserted. Because of this addition, FMN joined as amember of electron ﬂow. Domains of P450 and
NO_synthase that have binding sites for heme wereinserted on the lineages to type 2 and type 3 proteins, respectively. Types 2 and 3 proteins thus
acquired the ability to bind heme. On the lineages
to types 6 and 8, 2Fe2S ferredoxin was added by

174

the insertion of the Fer2 domain, which has 2Fe2S
ferredoxin binding sites. FAD was replaced by
FMN in type 8, although the FAD_binding_6 domain, which has the binding region of FAD or
FMN, did not change. Heme was also added on
the lineage to type 7 proteins because of the insertion of the Globin domain, which has heme
binding sites.
Lineages to types 9 and 10 proteins are more
complicated. Based on the phylogenetic tree in
Fig. 8D, we hypothesized that the insertion of thethree domains (Oxidored_molyb, Mo-co_dimer, andHeme_1) had occurred only once. The three domains
were then deleted three times on the lineage to
MCR1_YEAST, the lineage to NC5R_YEAST, andthe lineage to NC5R_BOVIN and NC5R_RAT independently. But the branching pattern of types 9
and 10 is not supported by high bootstrap probabilities. Therefore, we cannot predict how these proteins
were evolved in detail. In Fig. 9C, we illustrate thelineages of type 9 and 10 proteins that we hypothesized based on Fig. 8D.
The common features of the group 3 proteins arethat all proteins have the NAD_binding_1 domain.
NAD(P)H is their electron donor or acceptor, and
FAD or FMN is bound to them as an electrontransfer component. Although these features areconserved, electron transfer patterns in group 3 proteins are so diverged by adding and deleting various
domains.

Evolution of the Metabolism Systems
The four energy metabolisms and dissimilation in
sulfur metabolism are energy generation metabolisms. Proteins which can generate proton potential(abbreviations of such proteins are underlined inFig. 1) are conserved between diﬀerent metabolism
systems. Proton potential is the source for the synthesis of ATP by ATP synthase. This function isimportant for energy generation metabolisms. Proteins which have this important function, generationof proton potential, seem to be conserved among thefour energy metabolisms and dissimilation in sulfur
metabolism. This suggests that those systems are evolutionary related.
We therefore tried to infer the evolutionary relationship of the four energy metabolisms and dissimilation metabolism. Trees A–E in Fig. 10 show theﬁve phylogenetic trees of homologous proteins analyzed in this study and previous analyses (Berry et al.
2000; Saraste et al. 1994). Divergence between eubacteria and others (archaea and eukaryotes) are
shown in Fig. 10 based on rough estimations of thisstudy (Figs. 3, 5, and 7) and previous studies (Berryet al. 2000; Saraste and Castresana 1994).

Those protein trees were superimposed to infer theevolutionary tree of metabolic systems. The idea ofsuperimposition came from Oota and Saitou (1999),
who inferred the evolutionary tree of muscle tissues
by superimposition of muscle protein trees. Because
the protein cluster of denitriﬁcation (NDN), sulfur
respiration (SR), and dissimilation in sulfur metabolism (SDS) was estimated to emerge after the divergence of eubacteria and archaea/eukaryotes
according to tree A, these three systems were considered to be closely related. Therefore, NDN of trees
B and C in Fig. 10 were assumed to correspond to
the NDN –SR –SDS cluster of tree A. Trees A, B, andC in Fig. 10 were thus superimposed to produce treeF.
We then superimposed trees D and E in Fig. 10.
Because our interest is the order of system generation, we ignored the newer lineage of nitrate assimilation (NAS) and assimilation in sulfur metabolism(SAS) in these two trees. The resultant superimposed
tree is tree G in Fig. 10. Trees F and G were further
superimposed to produce single phylogenetic tree Hof the seven metabolic systems. However, because of
the lack of information, the branching point of the
aerobic respiration system was not able to be determined, and there are ﬁve branching possibilities(1–5 in tree H, Fig. 10). Tree H suggests that photosynthesis is basal (diverged ﬁrst) among the energy
metabolisms we analyzed. Aerobic respiration might
be utilized after the increase in oxygen by photosynthesis, while ancestral sulfur and/or nitrogenmetabolism system coexisted with that. Therefore,
we considered that branching position 1 may bemost plausible among the ﬁve possibilities for the
emergence of the aerobic respiration system (see tree
H in Fig. 10). In fact, a recent study suggested thatthe atmospheric oxygen was present at signiﬁcantlevels around 2.3 billion years ago (Bekker et al.
2004).
If we accept tree H, the assimilation process (nitrate assimilation and assimilation in sulfur metabolism) seems to be older than the dissimilation process(denitriﬁcation, sulfur respiration, and dissimilationin sulfur metabolism). Assimilation systems might begenerated before the divergence of eubacteria andarchaea/eukaryotes, and dissimilation systems might
be generated after the divergence of eubacteria andarchaea/eukaryotes.
Although tree H does not show the ancestral system at the branching nodes, we can infer that nitrateassimilation was ancestral to the sulfur assimilation
metabolism. Nitrate assimilation is involved in themetabolism for producing any amino acids, while
assimilation in sulfur metabolism is involved only for
producing amino acids including sulfur (cysteine andmethionine). It is conceivable that generation of nitrate assimilation enabled ancient organisms to pro-

175

duce amino acids except for cysteine and methionine
at ﬁrst, followed by acquisition of the ability for
producing cysteine and methionine by assimilation in
sulfur metabolism.
The above conjectures were based on the superimposed tree H in Fig. 10. Because this tree wasproduced through various assumptions, some or allof our conjectures may turn out to be incorrect.
Nonetheless, we would like to point out the possibility that the phylogenetic tree of proteins can infer
the phylogenetic relationship of metabolism systems.
Acknowledgments.
We are grateful to Dr. Hiromi Suzuki of
Meiji University for helping us in the initial part of this study. We
appreciate Dr. Takeshi Kawabata of NAIST for advising us of the
PSI-BLAST homology search method and helping us to conduct it.
Color version of all ﬁgures is available at http://sayer.lab.nig.ac.jp/
tomiki/JME_metabolism/.

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