FEMS Microbiology Letters 122 (1994) 203-210
© 1994 Federation of European Microbiological Societies 0378-1097/94/$07.00
Published by Elsevier
203
FEMSLE 06190
MiniReview
Membrane-bound Bacillus cytochromes c
and their phylogenetic position among bacterial
Class I cytochromes c
Nobuhito
Sone * and Hiroyuki Toh
Department of Biochemical Engineering and Science, Kyushu Institute of Technology, 680 Kawazu, lizuka, Fukuoka-ken 820, Japan
(Received 5 May 1994; revision received 21 July 1994; accepted 3 August 1994)
Abstract: Gram-positive bacteria lack a periplasmic compartment and contain only membrane-bound cytochromes c. There are at
least two types. One is found in subunit II of cytochrome oxidase, and the other is small cytochrome c which is also
membrane-bound because of an unprocessed signal sequence or post-translational acylation at the N-terminal end of the protein.
These Bacillus cytochromes c are compared with known class I cytochromes c, and a phylogenetic tree has been constructed by the
neighbour-joining method.
Key words: C~tochrome c; Bacterial respiration; Bacillus; Dendrogram
Introduction
L i t t l e is k n o w n a b o u t c - t y p e c y t o c h r o m e s of
G r a m - p o s i t i v e b a c t e r i a r e l a t i v e to t h o s e o f
G r a m - n e g a t i v e b a c t e r i a . This is p a r t l y b e c a u s e in
Gram-positive bacteria c-type cytochromes are
m e m b r a n e - b o u n d a n d t h u s m o r e difficult to purify t h a n t h e i r s o l u b l e c o u n t e r p a r t s in G r a m n e g a t i v e b a c t e r i a . R e c e n t studies have r e v e a l e d
t h e p r e s e n c e a n d c h a r a c t e r i s t i c s of several c - t y p e
c y t o c h r o m e s in G r a m - p o s i t i v e b a c t e r i a , especially
in the g e n u s Bacillus ( i l l u s t r a t e d s c h e m a t i c a l l y in
Fig. 1). In this review we will d e s c r i b e s t r u c t u r a l
* Corresponding author. Fax: (+ 81-948) 29 7801.
SSDI0378-1097(94)00342-4
c h a r a c t e r i s t i c s of Bacillus c y t o c h r o m e s c a n d
c o m p a r e t h e m with o t h e r b a c t e r i a l Class 1 cyt o c h r o m e s c, which a r e small, m o n o - h e m e cyt o c h r o m e s with the h e m e C covalently b o u n d
close to the N - t e r m i n u s [1].
Bacillus cytochrome c fused with cytochrome oxidase
O n e Class I c y t o c h r o m e c is p r e s e n t in the
c y t o c h r o m e caa 3 oxidase of Bacillus species. T h e
t h e r m o p h i l i c b a c t e r i u m PS3, a strict a e r o b e isol a t e d from a hot s p r i n g in J a p a n a n d similar to B.
stearothermophilus, has a r e s p i r a t o r y c h a i n composed of primary dehydrogenases, menaquinone-
204
7, c y t o c h r o m e bc x complex and cytochrome c
oxidase at least w h e n the ceils are cultured u n d e r
highly-aerobic conditions [2,3]. T h e c y t o c h r o m e c
oxidase is c o m p o s e d of four subunits [4] and has
four redox centres called c y t o c h r o m e a, cytochrom e a 3, Cu A and Cu~ [3,5]. A characteristic feature of this oxidase is the presence of covalently
b o u n d h e m e C, which is b o u n d to subunit II
together with the Cu A centre [5,6]. W e class this
kind of c y t o c h r o m e c as IIc. Cloning and sequencing of the caa (or cta) o p e r o n encoding the
oxidase subunits have shown that the heine of
this cytochrome c is b o u n d at the distal end of
subunit II [7]. T h e c y t o c h r o m e c d o m a i n is composed of about 9 0 - 1 0 0 residues. T h e overall sequence of this bacterial cytochrome c is dissimilar to mitochondrial c y t o c h r o m e c, but it is still
lysine-rich and several residues are conserved.
This I l c seems to be analogous to the mitochondrial c y t o c h r o m e c b o u n d at the high-affinity site
of c y t o c h r o m e c oxidase [6], since the super complex of the bc 1 complex and cytochrome caa3-type
oxidase oxidized quinol at an appreciable rate
and without exogenous c y t o c h r o m e c [8,9]. Similar cytochromes caa 3 have also b e e n f o u n d in
other Bacillus species such as B. subtilis [10,11],
B. firmus O F 4 [12], B. stearothermophilus [13] and
B. cereus [14].
A
Small cytochrome c in Bacillus
A gel electrophoresis pattern of m e m b r a n e s of
Bacillus PS3 in the presence of sodium dodecyl-
sulfate is shown in Fig. 2. Four or five components are seen after heme-staining for cytoc h r o m e c. T h e 38-, 28- and 22-kDa c o m p o n e n t s
are the subunit II of cytochrome caa 3, the cyt o c h r o m e c 1 ( f ) and the c y t o c h r o m e b subunit of
the bcl(bf ) complex, respectively [5,8,9]. T h e
smallest c o m p o n e n t , about 10 kDa, corresponds
to a cytochrome c-551 [15]. T h e a m o u n t of this
small cytochrome c was m o r e a b u n d a n t in the
m e m b r a n e s of the cells cultured u n d e r air-limited
conditions (Lane 2). T h e similar cytochrome c
(c-550) is also f o u n d in B. subtilis m e m b r a n e and
was also induced by air-limitation as reported by
V o n W a c h e n f e l d t and H e d e r s t e d t [16,17]. A similar pattern of m e m b r a n e - b o u n d cytochromes c is
also observed in B. subtilis m e m b r a n e s [17]. T h e
gene (cccA) coding for the B. subtilis 13-kDa
c y t o c h r o m e c-550 (121 amino acids) has been
cloned. Analysis of the m a t u r e protein has shown
that its signal peptide is not processed and functions to a n c h o r the cytochrome to the m e m b r a n e
[16]. T h e small cytochrome c of Bacillus PS3
m e m b r a n e s can be extracted with a mild surfactant such as cholate. It has been purified and
B
300H
.
.
.
C
COOH ...~_OOH
.
- - - - ~ , r
t,
NH 2
_
_
_
NH2
-
.
.
.
.
.
~H2
Fig. 1. Schematic representations of the three types of membrane-bound Bacillus small cytochromes c. (A) Cytochrome caa3 where
cytochrome c is a part of subunit II of cytochrome oxidase as a result of gene fusion. The chromophores of cytochrome c oxidase
such as Cu A in subunit II, and Cu B, a, and a 3 in subunit I, as well as c, heme C of cytochrome c moiety, are shown. (B) Bacillus
subtilis c-550 with the signal sequence as a membrane anchor. (C) The signal peptide of Bacillus PS3 c-551 which is processed, and
diacyl glycerol residues locate the protein in the membrane.
205
A
Mr
B
coxl
coxll
C1
b
C-551
Fig. 2. SDS gel electrophoresis of membrane fraction of
thermophilic Bacillus PS3, showing polypeptides containing
heme. A, membranes from vigorously aerated cells; B, membranes from air-limited cells. Mr, the Bio-Rad pre-stained
marker-proteins (106, 75, 49, 32, 27, 17 kDa. Coxl and coxlI
are the largest and second largest subunits of cytochrome c
oxidase, while c~ and b are the cytochrome subunits of
cytochrome bct(bf) complex.
named PS3 c-551 [15]. The structural gene for
this cytochrome has been cloned and sequenced
[18]. Lipase treatment, reverse-phase chromatography, protein sequencing, and ion-spray mass
spectroscopy have revealed that the mature cytochrome c-551 has a diacylglycerol attached via
a thioether bridge at the N-terminal cysteine (the
19th amino acid residue of the nascent peptide)
[I8]. Lipoproteins such as the cytochrome c subunit of the Rhodobacter L,iridis reaction centre
and the Escherichia coli major outer membrane
protein are known to have the N-terminal cysteine residue modified with diacylglycerol [19].
The N-terminal cysteines of these proteins are
known to have amino groups, but the N-terminal
cysteine of PS3 c-551 is blocked [18]. A plausible
structure of PS3 c-551 as a lipoprotein is shown
in Fig. 3. Thus, at least three methods are possible to make small Bacillus cytochromes c membrane-bound (see also Fig. 1).
In addition to Bacillus PS3 and B. subtilis, the
partial amino acid sequences of B. lichenformis
and B. azotoformans cytochromes c have been
reported [20]. These three sequences of small
Bacillus cytochromes have been compared with
those of Pseudomonas aeruginosa c-551 (c 8) and
Anacystis nidulans c 6. The Bacillus sequences
show common features with both c 6 and c8, but
are at the same time clearly different from both
types, indicating that they may form a new group
of class I cytochromes c [18].
Several small 'soluble' Bacillus cytochromes c
have been purified; c-551 and c-554 from B.
subtilis [21], c-550 from thermophilic Bacillus PS3
[22], c-552 from alkaliphilic B. firmus RAB [23],
and c-551, c-552 and c-554 from B. licheniformis
[24]. These cytochromes are probably derived
from membrane-bound forms by proteolysis. Soluble fractions of Bacillus PS3 [15], B. subtilis [16]
and alkaliphilic Bacillus YN-2000 [25] contain no
cytochromes. C-type cytochromes generally function in the periplasm or the periplasmic surface
of the inner membrane. In this respect it is noteworthy that Hreggvidsson solubilized c-552 and
c-555 from membranes of B. azotoformans by the
addition of trypsin [20]. The same thing also
occurs with PS3 membranes (Sone, unpublished).
The role of these small Bacillus cytochromes c
has not been elucidated. Von Wachsenfeldt and
CH20-CO-C•sH3•
CHO-CO-CIsH31
CHacO- Nit- C 19. . . . . . . . . . . . . . .
C-A-S-C-H .......
A_K_K 111COOil
Fig. 3. A likely structure of PS3 cytochrome c-551 containing diaeyl glycerol [18].
206
A
I
.........
+ .........
+ .........
+ .........
+ .........
+ .........
+ .........
+ .........
+ .........
+ .........
+ ........
I ) ~QLAKQKGCMACHDLKAK
.... KVGP-AYADVAKKYA
.................
-GRKDA---VD-YLAGKIKKGGSNGVW
2)
GEALFKSKPCAACHSIDAK
.... LVGP-AFKEVAAKYA
.................
-GQDGA-
3 ) GEELFKSKPCGACHSVQAK
.... LVGP-ALKDVAAKNA
.................
-GVDGA---AD-VLAGHIKNGS-TGVW
......
-C~MP-PNP--VT--EEEAKTLAEWVLTL
4 ) PEVLFKNKGCVACHAIDTK
.... MVGP-AYKDVAAKFA
.................
-GQAGA---EA-ELAQRIKNGS-QGVW
......
-GPIPMP-PNA--VS--DDEAQTLAKWVLSQ
- -AD-LLAGH
.......
+ .........
IKNGS -QGVW .......
GSVPMP-PQN--VT--DAEAKQLAQWILSI
GP IPMP-PNP
--VT--EEEAK
I LAEWILSQ
5 ) GEELYKTKGCTVCHAIDSK
.... LVGP-SFKEVTAKYA
.................
-C-QAGI---AD-TLAAKIKAC-G-SGNW
......
-GQIPM~-PNP--VS--EAEAKTLAEWVLTH
6 ) AKALAQKSGCLACHSIDAK
.... VLGP-AYKDVAAKYK
.................
-GDKGA---EA-KLIEKVKKGG-SGVW
......
-GNIPMP-ANSPQVK--DEDIKTIVEWILTL
.................
-GQAGA---PA-LMAERVRKGS-YGIF
......
-GYJ~PMT-PTPPARI-SDADLKLVIDWILKT
7
) PAELATKAGCAVCHQPTAK----GLGP-SYQEIAKKYK
8)
AEQ I F -KQNCASCHGQDLSG--
-GVGP -NLQKVGSKYS
..........................
KD-E IKNI IAN .............
9}
GEE I Y -QQNC TGC HG~DLAG--
-GSAP - S LKEVGGKYK
..........................
ES-E IKDIVVN
..............
GRGGMP
- - -GN-LV
10)
PEE I Y -KANC IACBG~
-VSGP -S LKGVGDKXD
..........................
VA-E IKTKIEK
..............
GGNGMP
- - - SG-LV -PADKLDDMAEWVSK
11)
GEDIV-GKTCNTCHGTGL
NYEG--
12 ) A D D I I - A K H C N A C H C ~ K G V
13)
14)
..... LGAP-KVGI~KAF
.....................
WGKRAE
..... LGAP-KIGDTAA
.....................
WKERAD--HQGGLDGILAKAI-S
.....................
.....................
WE PRM- - -AK -CAMDSLVQSVK-N
.........
-GLNAMP
W A P H I - - - A K - ~ S
I-KGYKG ...... TKGMMP
GEAVY-NKACQTCHAMGI
..... AGAP-ELNDAAA
GKATY-DASCAMC
BKTC44 ..... MGAP-KVGDY~AA
- -EQGGLDGLLAKAI
-GRGAMP
-S .........
.... VV~A--AKTL-KKED-
.................
LVKYG---KD-SVE-AIV-TQ-VTK
........
.... VV~P--AKTL-EKAD.... VVMA--NKTL-KKEA
.................
..................
LDEYG---MA-SIE-AIT-TQ-VTN
LEQFG---MN-SAD-AIM-YQ-V~N
.......
.......
18 ) GASVF-SANCAACH~
.... VIVA--NKTL-SKSDLAK
19 ) GEKVF-SANCAACHAGGNN
.... AIMP--DKTL-KKDV
.......
-G~NAMP-GFNGRLS--PLQIEDVAAYVVDQ
LEANS---MN-TID-AIT-YQ-VQN
.......
- G ~ - A F G
..................
LTANG---KD-TVE-AIV-AQ-VTN
.......
-G~GAMP-AFKGRLS--DDQIQSVALYVLDK
.......
-(~(GPMP -AWE GVLS - -E DE IVAVTDYVYTQ
.... VISA--GKVL-SKTAIE
...............
..............
23 ) GEQVF-TGNCAACBSVEEE
.... KTLE--LSSL-WKAK
FYLDGG--
-Y- -TKE -AIE -YQ-VRN
-QYLDC-G---F--KVE-SSI-YQ-VEN
.................
24 ) GAKIY--AQCAGCBQQNGQ-----GIPG-AFPPL-AGHVAEI
SYLANF---NG-DES-AIV-YQ-VTN
..............
........
.......
RLV--DEDIEDAANYVLSQ
(~(GAMP-AWADRLS--EEEIQAVAEYVFKQ
-GKNAMP-AFGGRLE--DDEIADVASYVLSK
LAKEGG---RE-YLI-LVLLYG-LQGQIEVKGMKYNGVMS-SFAQ-LK--DEEIAAVLNHIATA
25 ) GED~I--GTCVACHGTDGQ----GLAP-IYPNLTGQSA
.................
TYLESS---IKAYRDGQ-RKGG-NAA
26 ) GQAKA--AVCGACBG~DGN
.... SAAP-NFPKLAGQGE
.................
RYLLKQMQDIKAGTKPGAPEC-SGRKVL
27)
- -- -GNTPAAYPRLSGQHA
................
-QYVAKQLTDFREGARTNDGD
28 ) GKTLY-DASCASCBG~QAQ----GMFP-KLAGLTSE
29 ) GESLF-ATSCSGCHG~LAEG---KLGP----GLNDNYWTY
....................
.................
30 ) AEELY-AGMCSGCBG~YAEG---KIGP----GLNVAYWTY
.................
31) GAALY--KSCVGCHC4%DGS
.... KQAM-32 ) GEELFKEKNCLSCHAVEPND---K-RA-E
Gt4GAMP-AFGGRLS--AEDIEAVANYVLAQ
-G~GAMP-AFGAKLS--ADDIEGVASYALDQ
-(~KNAMP-AFGGRLS--EAQIENVAAYVLDQ
YLKGFD---DD-AVA-AVA-YQ-VTN
.... SVMP--EKTL-DKAALE
-PAC TGCHSPNGE
-PGGMC~C
S DDDYKAAIE FMAE K
-AKGGNPKLTDAQVGNAVAYMVGQ
..............
GADVF-ADNCSTCHVNGGN
........
.......
..............
124TPMA----QGLS--DEDIADIAAYYSSQ
EMTGML
.... DNFS--DQDLADLAAYFTSQ
t~4- I M R - S I A A K L S -
-NKDIAAI
SSY IQGL
RIKTT---L--VAFKSGDTAT-LKKEG-LGGP-NSAIMA-PNAAGLS--EQDMDNLSAYIATL
PSNT---TDVGLFATIF-GG...........
ANGMMGPHI~N-LT--PDEMLQTIAWIRHL
PGNE---TDVGLFSTLY-GG
--GVGHAVKGQ
................
KADE LFKKI/( -GYADGSY
.... AARTAPNL-A-TFGE-RTKV-AGVKEAN---KE-NV~PD-SIKP
............
ATGQMGPMWGS-LT--LDEMLRTMAWVRHL
-GG-E ..........
KKAVMT -NLVKRYS - - DEEMKAMADYMSK
...... -G-I~fGTYPK-LS--DSETNALYEYLKGL
33 ) G~AIF-NKSCIGcHAVTPLD---K-RP-A----QRRTAPNL-A-DFGD-RERI-AGILEHN---EE-NLKKWLRDPN-SVKP
......
-G-NKMA~GH-LT--EEQIDALTKYLMSL
34 ) GRQVFEENSCIGCHAVGGTGT--AA~-AF
............
......
-G-NVMPA-YPD-MS--EEDMEALIAYLRSL
35 ) GQ~-QQNCAACBGVARSMPPAVIGP-EL
. . . . . . . . . . .
......
-G-VKMPG-FPQ-LS--EEDLDALVRYLEGL
T-NFGE-REVI-AGYLENN---DE-NLEAWIRDPQ-SLKQ
-G-LWGN-RTSLGAGIVENT---PE-NLKAWIRDPA-C~
36 ) GATLFKTR-CLQCHTVEKGGPH-KVGP-NLHGIFGRHSGQAEGYSYTDANIK---KNVLWD---EN-NMSEYLTNPK-KYIP
37)
GKKTFVQK
-CADCHTVENGGKH
-KVGP -NLWGLFGRKTGQAEGYS
YTDA~
- - -KGIVWN-
......
- -ND -TLME YLENPK
38 ) G~J%AF-NK-CKACHEIG~SAKN-KVGP-ELNGLDGRHSGAVEGYAYSPAt~J%---SGITWT---EA-EFKEYIKDPK-AKVP
39 ) APPAFGM--CKAC~SVEAG-KN-GVG~-SLAGVYGRKAGTLAGFK~SDPHAK---SGLTWD---EP-TLTKYLADPK-GVIP
40)
GEKVSKK
- -C LAC B TFDQGC.AN-KVGP
O
-NLFGVFE
NTAAHKDNYAYSE
S YTEMKAKGL~%~T-
- -EA-NLAAYVKDPK
-G-TKMA--FGG-LK-KEKDRNDLITYLKKA
-Ky I p ......
-G-TKM
I --FAG-
IK -KKGERQDLVAYLKSA
......
-G-TKMV--FAG-IK-KDSELDNLWAYVSQF
......
-G-~J~V--FAG-LK-NPADVAAVIAYLKSL
- A F V L E ~ D P ~ T - - ~
OO
- - LT-~DE
o
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B
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Tne c 4
Pde CL
I
KHMSGL
..................
22 ) GAQVF-NGNCAACHMGGRN
LADGM-
KAA
K
GINAMP-PKGTCADCSDDELREAIQKMSGL
16 ) GGQ~-SANCASCBLGGRN
17 ) GAKVF-SANCAACHMGGGN
21)
- DE KEAEAVAKWLSE
-GINAMP-PKGTCADCSDDE
..........
15 ) GASIF-SANCASCHMGG~N
20 ) GGKVF-NANCA~CHASGGG----QING--AKTL-KKNA
- - -AG- I I -KGE DADKVAEWLAAK
,~6
0 25 33
PS3 IIc
asrt*,,,c
I
O2
i
IE ~ T L
L
207
H e d e r s t e d t d e l e t e d t h e c c c A g e n e o f B. subtilis
a n d f o u n d t h a t t h e m u t a n t cells grew as well as
the w i l d - t y p e [16]. T h e B a c i l l u s PS3 c-551 m a y b e
an e l e c t r o n d o n o r for an a l t e r n a t i v e o x i d a s e o t h e r
t h a n the c y t o c h r o m e c a a 3 - t y p e oxidase. T h e c-551
is p r o d u c e d u n d e r a i r - l i m i t e d c u l t u r e c o n d i t i o n s
w h e r e t h e a l t e r n a t i v e oxidase f u n c t i o n s [15], a n d
c y t o c h r o m e c is n o t n e c e s s a r y for quinol o x i d a s e
activity o f t h e s u p e r c o m p l e x c o m p o s e d o f t h e
c y t o c h r o m e b c a a n d c y t o c h r o m e c a a 3 - t y p e oxid a s e [8]. B. a z o t o f o r m a n s is k n o w n to p r o d u c e
c-552 a n d c-555 u n d e r b o t h a e r o b i c a n d denitrifying c o n d i t i o n s [20]. B. f i r m u s R A B c u l t u r e d at
a l k a l i n e p H p r o d u c e s c-552 u n d e r b o t h highly
a e r o b i c a n d a i r - l i m i t e d c o n d i t i o n s [23]. P e r h a p s
the role of t h e s e small c y t o c h r o m e s c will b e
clarified in t h e n e a r future.
Phylogenetic analysis of Class I cytochrome c
T h e two kinds o f B a c i l l u s c y t o c h r o m e s c, I I c
a n d small B a c i l l u s c y t o c h r o m e s c a r e a l i g n e d
with class I c y t o c h r o m e s c in Fig. 4A. T h e class I
c y t o c h r o m e s c, relatively small m o n o h e m e cyt o c h r o m e s with h e m e C in t h e N - t e r m i n a l part,
have b e e n s u b d i v i d e d into 1 A (long c2), 1B (middle c 2 a n d m i t o c h o n d r i a l c), 1C (split a - b a n d , c 6
a n d c a) 1D (c8), 1E ( % ) a n d o t h e r s [1,2]. D i c k e r -
son has a l r e a d y shown a l i g n m e n t s b a s e d o n t h e
t e r t i a r y s t r u c t u r e o f c y t o c h r o m e s c [26]. A n unrooted phylogenetic tree created by the
n e i g h b o r - j o i n i n g m e t h o d [27] is shown in Fig. 4B.
Class I c y t o c h r o m e s c i n c l u d i n g the two types
o f B a c i l l u s c y t o c h r o m e s c a r e classified into five
or six groups: (1) t h e c 2 a n d m i t o c h o n d r i a l cyt o c h r o m e c cluster, which also c o n t a i n s the B a c i l lus c y t o c h r o m e s c f o u n d in s u b u n i t II of t h e
c y t o c h r o m e c o x i d a s e ( I I c ) ; (2) the c 6 cluster; (3)
the s m a l l - s i z e d c y t o c h r o m e s c f r o m B a c i l l u s
which show r e l a t i o n s h i p with c6; (4) t h e r a t h e r
h o m o l o g o u s c 8 group; a n d (5) the fifth t y p e which
is very h e t e r o g e n e o u s c o n t a i n i n g c 4 (plus c L) a n d
c 5 groups. T h e l a t t e r g r o u p is k n o w n to c o n t a i n
e x t r a C y s - r e s i d u e s f o r m i n g a short disulfide l o o p
a n d m a y b e c o n s i d e r e d as a s u b - b r a n c h or t h e
sixth g r o u p [1]. D e s u l f o v i b r i o c,ulgaris c-553 a n d
Chlorobium lumicola (former thiosulfatephilum )
c-555 show w e a k similarity to this group, while
T h e r m u s t h e r m o p h i l u s c-552 [28] is i n c l u d e d in the
c 4 g r o u p with t h e f l a v o c y t o c h r o m e s [29]. T h e s e
t h r e e c y t o c h r o m e s , which a r e k n o w n to b e omitt e d f r o m any subclass [2], a r e all in t h e c 4 plus c 5
cluster. C y t o c h r o m e s c L of P a r a c o c c u s d e n i t r i f i c a n s a n d M e t h y l o b a c t e r i u m e x t o r q u e n s A M 1 form
a s u b - c l u s t e r s e p a r a t e d at a very early time. T h e
p a t t e r n s in the t r e e a r e c o n s i s t e n t with A m b l e r ' s
classification in g e n e r a l , e x c e p t t h a t he p l a c e s
Fig. 4. (A) Alignment of class I cytochromes c and (B) a phylogenetic tree for Class I cytochrome c including those from Bacillus.
(A) 1, Hydrogenobacter thermophilus c 8 (c-552)[c552-HYDTH]; 2, Pseudomonas stutzeri c8; 3, Pseudomonas mendocina Cs; 4,
Pseudomonas aeruginosa Cs; 5, Azotobacter vinelandii Cs; 6, Methylophilus methylotrophus c 8 [1]; 7, Rhodopseudomonas gelatinosa c 8
[1]; 8, PS3 c-551 [18]; 9, Bacillus licheniformis c-552 [20]; 10, Bacillus subtilis c-550 [16]; 11, Ps. aeruginosa c 5 [1]; 12, Ps. mendocina
Chll6 c 5 [30]; 13, Organism H-1-R c 5 [1]; 14, Chlorobium lumicola c-555; 15, Microcystis aeruginosa c 6 [CYC6-MICAE]; 16,
Anacystis nidulans c6; 17, Synechococcus sp. 6312 c 6 [CYC6-SYNSP]; 18, Spirulina maxima %; 19, Porphyra tenera %; 20,
Plectonema boryanum c 6 [CYC6-PLEBO]; 21, Euglena gracilis c6; 22, Chlamydomonas reinhardtii c 6 [CYC6-CHLRE]; 23,
Monochrysis lutheri c6; 24, Thermus thermophilus c-552 [28]; 25, Halophilic Micrococcus c-554; 26, Azotobacter L'inelandff c 4 (N side)
[29]; 27, A. L'inelandii c a (C side) [29]; 28, Thiobacillus neapolitanus c-554(548) [C554- THINE]; 29, Methylobacterium extorquens c k
[CYCL-METEX]; 30, P. denitrificans c L [CYCL-PARDE]; 31, Desulfouibrio c'ulgaris Miyazaki c-553 [C553- DESVM]; 32, B.
subtilis llc [10]; 33, Bacillus PS3 IIc [7]; 34, Bacillusfirmus Ilc [12]; 35, T. thermophilus IIc [34]; 36, Saccharomyces cereL,isiae c iso-1
[CYC1- YEAST]; 37, tuna c; 38, Nitrobacter winogradskii c 2 [C550- NITWI]; 39, Rhodospirillum fulvum c2, iso-1; 40, Rhodospirillum rubrum c 2. Extreme N-terminal and C-terminal parts are omitted from the alignment. The sequences without citation or
accession number in the amino acid sequence data base Swiss Prot are obtained from Dickerson's alignment [26]. Since the high
sequence divergence within the family prevented us from constructing a multiple alignment automatically, we used the alignment
which Dickerson previously constructed following the tertiary structures of the enzymes [26] when available. The new sequences
were added to the alignment after comparison using the Needleman-Wunsh method [35]. The positions of gaps were adjusted
consistent with those of Dickerson' alignment. (B) The phylogenetic tree was constructed by the neighbour-joining method [27].
The genetic distance of each aligned pair was evaluated as the number of amino acid replacements per site with Poisson correction.
A continuous gap in the alignment was treated as a single substitution.
208
both c 4 and c 6 which have a split a - b a n d in Class
1C [1]. Although both have the spectrally split
a-band, c 4 is quite different from c 6. The small
Bacillus cytochromes c are related to c 6 in the
tree. This result is consistent with the analysis of
16S r R N A [32] which shows relatedness between
cyanobacteria and Gram-positive bacteria. Also
the structure of the Bacillus cytochrome bcl(bf )
complex is rather similar to that of the cyanobacterial enzyme [9]. The sequences of Bacillus small
cytochrome c are, however, also similar to cs, as
noted before [16,23]. It is thus likely that the
small Bacillus cytochromes c may be a new group
in Class I cytochromes c. However, only two full
and two partial sequences are available to date.
Although this phylogenetic tree is unrooted, and
thus gives no definite information on the ancestral cytochrome, it is tempting to speculate that
the differentiation of Class I cytochromes c into
five (or six) subdivisions occurred at a very early
stage in the cytochrome c history. Dayhoff has
shown an evolutionary tree of Class Ic-type cytochromes, in which c 6 evolves from an ancestral
cytochrome c of an anaerobe, and c 2 and c s from
a primitive cytochrome c 6 (Fig. 4 in [31]). Since
many present bacteria are known to have two to
four kinds of Class I c-type cytochromes, it is
likely that differentiation for their functional roles
should be emphasized.
Roles of Class I cytochrome c
The functional roles of Class I cytochromes
have not been well documented except those of
c 2 and %, which provide electrons to h e m e / C u type oxidase as well as mediate electron transfer
between the bcl(bf ) complex and the photosynthetic reaction centre or photosystem I. Table 1
summarizes the roles (sometimes though putative) of the respective group of Class l cytochromes c as well as their structural characteristics. Since the physiological electron donors and
acceptors of each of the cytochromes c have been
determined rarely, the Table is rather speculative. However, we hope that our suggestions may
stimulate interest in determining their functions.
It seems likely that cs, including c5, is the substrate for an alternative terminal reductase such
as nitrite reductase. On the other hand, c 4 seems
to be an electron acceptor from dehydrogenases
which transfer electrons to the cytochrome corn-
Table 1
Characteristics of the six subdivisions of Class I cytochromes c
Phylogenic subclass
Electron transfer
group No.
Name
Donor
Acceptor
2.
I A + IB
( L c 2 X M c z)
IIc
IC (%)
bc 1
bc 1
/~f
b R C (PSI)
cyt.ox (aa 3)
cyt.ox (aa 3)
PSI cyt.ox
3.
IC (c 4)
DH
cyt.c
4.
IE (c 5)
As Cs?
as cs?
5.
ID (cs)
,7
Reductases
6.
Bacillus small c
bc i
Alternative
oxidase?
Structural
feature
Fused with COIl
Split a-band
M--LS--I--Y ~o
Split a-band,
aromatic and P after
heine site
Extra C, no aromatic near C-terminus
Several P after
W near C-terminus
With signal sequence
Main presence
Purple bacteria
Bacillus
Cyanobacteria,
chloroplasts
Nitrogen fixers,
Denitrifiers
Denitrifiers.
purple bacteria
Bacillus
bRC, bacterial reaction centre; PS, photosystem; COII, subunit 2 of cytochrome oxidase; DH, dehydrogenases; IA-E, the name of
subdivisions after Ambler [1]; M, medium; L, large.
a This motif is conserved among this group, but occurs more or less frequently in Class 1 cytochromes c belonging to other groups
as noted by Ambler [1].
209
ponent of the main electron transfer system. We
also propose that the thermophilic Bacillus cytochrome c-551 may function as substrate for an
alternative terminal oxidase other than the
h e m e / C u - t y p e oxidase [15]. In this respesct it is
noteworthy that a terminal oxidase functioning in
micro-aerobic soybean root nodules has a cytochrome with two C hemes, whose sequence is
rather similar to c 6 [32]. Saraste and Castresana
pointed out that this alternative oxidase may be
related to a primordial oxidase, and that it is
distant from other oxidases but still belongs to
the cytochrome oxidase super-family [33].
3
4
5
6
7
Conclusion
Having no periplasmic space, Gram-positive
bacteria contain m e m b r a n e - b o u n d Class I cytochromes c. One type of Bacillus cytochrome c
is found in subunit II of the aa3-type cytochrome
oxidase as a result of gene fusion. Another type,
the small cytochrome c, is m e m b r a n e - b o u n d via a
N-terminal hydrophobic polypeptide (signal sequence), or a diacyl glycerol moiety. The cytochrome c-moiety of the former type forms a
sub-cluster of the c 2 group, while the latter shows
a weak relationship to the c 6 group. The Class I
cytochromes c are divided into at least the following five branches; c2, c6, c8, c 4 plus c5, and
Bacillus small c. Differentiation of the ancestral
cytochrome c into these groups may depend on
its role in the electron transfer system.
Acknowledgements
We thank
Wachenfeldt
and valuable
review could
Prof. L. H e d e r s t e d t and Dr. C. von
for critical reading of the manuscript
comments. Without their help this
not have been realized.
8
9
10
11
12
13
14
15
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