(Received for publication, November 13, 1995; and in revised form, February 29, 1996)
From the
The qcr of Bacillus stearothermophilus K1041
encoding three subunits of the quinol-cytochrome c oxidoreductase (cytochrome reductase, bc
complex) was cloned and
sequenced. The gene (qcrA) for a Rieske FeS protein of 19,144
Da with 169 amino acid residues, and the gene (qcrC) for
cytochrome c
of 27,342 Da with 250 amino acid
residues were found at adjacent upstream and downstream sides of the
previously reported qcrB (petB) for cytochrome b
of subunit 25,425 Da with 224 residues (Sone,
N., Sawa, G., Sone, T., and Noguchi, S.(1995) J. Biol. Chem. 270, 10612-10617). The three structural genes for
thermophilic Bacillus cytochrome reductase form a
transcriptional unit.
In the deduced amino acid sequence for the FeS
protein, the domain including four cysteines and two histidines binding
the 2Fe-2S cluster was conserved. Its N-terminal part more closely
resembled the cyanobacteria-plastid type than the
proteobacteria-mitochondria type when their sequences were compared.
The amino acid sequence of cytochrome c was not
similar to either type; the thermophilic Bacillus cytochrome c
is composed of an N-terminal part corresponding
to subunit IV with three membrane-spanning segments, and a C-terminal
part of cytochrome c reminiscent of cytochrome c-551
of thermophilic Bacillus. The subunit IV in the enzyme of
cyanobacteria and plastids is the counterpart of C-terminal part of
cytochrome b of proteobacteria and mitochondria. These
characteristics indicate that Bacillus cytochrome b
c
complex is unique.
Thermophilic bacilli have menaquinol-cytochrome c oxidoreductase (cytochrome reductase, cytochrome bc
complex) and caa
-type cytochrome c oxidase as major
respiratory
complexes(1, 2, 3, 4, 5) ,
while aa
-type menaquinol oxidase is known to
oxidize menaquinol in mesophilic bacilli such as Bacillus subtilis(6, 7, 8, 9) and Bacillus
cereus(10) . Cytochrome reductase purified from Bacillus PS3, a thermophilic bacterium isolated from a hot
spring in Japan closely related to Bacillus
stearothermophilus, was found to contain four chromophores in four
subunits- 29-kDa cytochrome c
, 23-kDa Rieske
iron-sulfur protein, 21-kDa double-heme cytochrome b
, and 14-kDa subunit IV (1, 2) .
Cytochrome b/b + subunit IV with two low
spin protohemes play important roles in generating proton motive force
by the quinone cycle in the reductase(11, 12) .
Cytochrome b
, with two protohemes of the
cyanobacterial-plastidal counterparts of cytochrome b of about
420 aa, (
)is known to be homologous to the N-terminal half
of cytochrome b of proteobacteria and mitochondria. Subunit IV
of about 160 aa is homologous to the C-terminal half of cytochrome b(13, 14, 15, 16) . The FeS
protein and cytochrome c
/f are known to
be simple electron donors to cytochrome c.
Proteobacterial-mitochondrial cytochrome c
uses
His and Met as axial ligands for heme, while cyanobacterial-plastidal
cytochrome f uses His and Tyr(17) ; these two
cytochromes exhibit almost no sequence homology(15) . As for
the Rieske FeS protein, the C-terminal halves are conserved, while the
N-terminal halves are not(15) .
We cloned the cytochrome b locus from transformable B. stearothermophilus K1041 (Bst) and found that the gene for cytochrome b encodes a 224-aa protein with four hydrophobic
helices possessing two pairs of His as ligands for the two protoheme,
while the gene for subunit IV corresponds to the C-terminal half of
cytochrome b(18) . The two genes were adjacent, but
separated, indicating that the Bst locus is rather similar to b
subunit IV gene organization in cyanobacteria.
In addition, several features of deduced amino acid sequences also
support evolutionary relatedness to cyanobacterial cytochrome b
. 1) The two His in the fourth transmembrane
segment are separated by 14 aa residues as in cytochrome b
, not 13 aa as in the proteobacterial and
mitochondrial cytochrome b; 2) His
of yeast
cytochrome b, which may constitute a part of the Qi site, is
replaced by Arg as in cytochrome b
; 3) Lys
(as defined in the yeast sequence) is conserved among antimycin
A-susceptible forms of the cytochrome but is replaced by Asn as in
cytochrome b
. We found later, however, that the
postulated gene for subunit IV downstream from cytochrome b,
did not stop after 173 aa residues(18) .
The genes for three
subunits of the bc complex form the fbc operon in proteobacteria(20, 21) , while the
genes (petBD) for b
and subunit IV sit
apart from petCD for the FeS protein and cytochrome f in cyanobacteria(14, 22) . A recent paper on Chlorobium limicola showed a third case in which the genes for
the FeS protein and cytochrome b are linked, but the gene for
cytochrome c
is not found downstream of the gene
for cytochrome b(23) . Nonetheless, the deduced amino
acid sequences of the two C. limicola subunits showed close
similarity to the cyanobacterial subunits.
By analyzing upstream and
downstream regions of the gene for Bst cytochrome b, the genes for FeS protein and cytochrome c
are found to form a qcr operon as in
proteobacteria. However, the sequence of Bst cytochrome c
is quite different from that of purple bacterial
cytochrome c
; the Bst c
is
composed of a membrane domain homologous to subunit IV and a
hydrophilic part homologous to Bacillus small cytochrome c ( (24) and (25) ; see also (26) for
review).
Figure 1:
A map
of Bst DNA around the qcrABC operon encoding the FeS,
cytochrome b, and cytochrome c
, and sequence strategy. Three clones, two new
clones (pFBCX3 and pFBCSS16) in addition to bcl5 (SalI-EcoRI fragment) as described
previously(18) , cover the whole operon. Putative promoter
(
) and terminator (
) regions, and primers (
)
used for PCR are shown.
Fig. 2shows typical results of Northern blotting with Bst RNA. Both qrcB and qrcC probes hybridized to main bands at 2.1 kilobases, indicating that the putative promoter and terminator (Fig. 3) are probably assigned correctly, and the transcript is polycistronic for three subunits of the Bst cytochrome reductase. The radioactive bands of a high molecular weight seem to be due to contamination of genomic DNA.
Figure 2: Northern blot analysis of Bst RNA with qcrA and qcrC as the probes. RNA loadings were 5 µg (lanes 1 and 4), 10 µg (lanes 2 and 5), and 20 µg (lanes 3 and 6). Lanes 1-3, probed with qcrB. Lanes 4-6 probed with qcrC. The positions of RNA size standards are as indicated. Exposure time was 12 h.
Figure 3:
DNA and deduced amino acid sequences of Bst FeS protein, cytochrome b, and
cytochrome c
. Putative Shine-Dalgarno sequences
are boxed. The nucleotides that may constitute the putative
promoter region are boxed and shaded. The arrows indicate where putative stem-loop structure might form the
terminator of the gene. The residues from N-terminal sequence analyses
are shown in boldface letters.
Figure 4: Multiple alignment of the Bst FeS protein with those from various sources. The proteins from rat mitochondria(37) , yeast mitochondria(38) , R. sphaeroides (Rs, (39) ), spinach plastids (Spi; (40) ), Synechocytosis PCC6803 (Sy; (41) ), Nostoc PCC7906 (No; (22) ), and C. limicola(23) are compared with the Bst protein. The conserved FeS-binding motifs are boxed, and all residues identical with those of Bst are shaded.
Figure 5:
Hydropathy profiles of Bst FeS
protein (A) and cytochrome c (B). The procedure of Kyte and Doolittle (36) was used
with a window of 15 residues. The FeS-binding sites (cf.Fig. 4) in A are hatched. The arrows in B show heme C-binding
site.
Figure 6:
Multiple alignment of Bst cytochrome c with subunit IVs (A)
and Bacillus small cytochromes c (B). The
N-terminal part of Bst cytochrome c
is
compared with subunit IVs of Nostoc(22) and maize
plastids(19) . The C-terminal part is aligned with PS3
cytochrome c-551(25) , B. subtilis
c-550(24) , and B. licheniformis
c-552(30) . Heme C motifs are boxed, and residues
identical with those of Bst cytochrome c
are shaded.
The presented data demonstrate that the structural genes for
three subunits of thermophilic Bacillus cytochrome reductase
constitute an operon structure like that of the proteobacterial fbc operon. However, the Bacillus cytochrome b is small, and its sequence is most closely related to cytochrome b
of cyanobacteria as reported
previously(1, 2, 18) . The petB gene
for cytochrome b
of cyanobacteria is followed by petD for subunit IV, which has sequence homology with the
C-terminal half of cytochrome b of
proteobacteria(14, 15) . We reported that Bst cytochrome b
was small due to the presence of
a stop codon at position 225 leaving the subunit IV part to be encoded
on another gene as in cyanobacteria (18) . At first we thought
that the Bst subunit IV is equivalent to cyanobacterial
subunit IV, since a stop codon for the presumed Bst subunit IV
was found at a similar place. The stop codon, however, turned out to be
the result of a frameshift due to a misread of the DNA
sequence(18) . We previously cloned the PS3 cytochrome b
gene by PCR using N-terminal sequences of
cytochrome b
and subunit IV for designing a set of
primers targeting WRDIAD and MKFENT, respectively (18) . The
deduced aa residues of qcrC, presumably encoding subunit IV,
coincided with the N-terminal sequence only at the first three residues
(MKF), but the subsequent residues were totally different, even though
the clone (bc15) was obtained by using the probe prepared by
PCR(18) . We also determined that the N-terminal sequence of
PS3 cytochrome c
is MHRGLGMKFV- (Table 1), and found that this corresponds to the ORF previously
ascribed for ``subunit IV''(18) , when translational
initiation occurs 6 aa residues further upstream. Moreover, there is a
heme C-binding motif, CXXCH, at about 580 bp downstream of the
initiation codon. Cytochrome c
, which is
SDS-dissociated and fractionated by reverse-phase high pressure liquid
chromatography, possesses a heme content of 32 nmol/mg of protein,
indicating that the heme-staining protein of about 30 kDa has one heme
C. (
)The qcrC gene encodes a 27,342-Da protein with
250 residues, suggesting that cytochrome c
retains
subunit IV as an integral N-terminal component. We were also able to
prepare and sequence peptides possessing IAQANTXTSXHGENL- by trypsin
digestion, and PGGIFKGTDEELQK- by treatment with cyanogen
bromide, showing directly that the deduced aa sequence of the c-type cytochrome found in PS3 29-kDa protein is represented
in the sequence for Bst qcrC.
What, then, is the identity
of the ``band IV protein'' that copurified with cytochrome
reductase from the thermophilic Bacillus PS3(2) ? Much
of the band IV protein in the PS3 enzyme preparations can be removed
without severe loss of quinol-dependent cytochrome c reductase
activity by gel filtration in the presence of lauroyl
sarcosinate. Thus, this protein may be contaminant or an
auxiliary component of the enzyme. On the other hand, it is also
noteworthy that some purple bacteria such as Rb. sphaeroides are known to contain subunit IV of 14 kDa(31) , while Rb. capsulatus contains three subunits(32) .
The
bacterial cytochrome reductases have been divided into two subclasses; bc-type is found in proteobacteria and
mitochondria, while the b
f-type occurs in
cyanobacteria and plastids having b
and the
subunit IV instead cytochrome b(13, 14, 15) . The three subunits
of bc
-type are encoded in qcrABC operon,
while petBD for cytochrome b
plus subunit IV and petCA for the FeS protein plus cytochrome f are at separate sites in cyanobacterial
genomes(14, 22) . It is also pointed out (15) that the N-terminal halves of the FeS proteins and
cytochrome c
and cytochrome f are not
homologous, suggesting their different evolutionary origins for
proteobacteria and cyanobacteria. Bacillus cytochrome
reductase is apparently a third type, different from both
proteobacterial and cyanobacterial counterparts. The structural genes
for cytochrome reductase of the thermophilic Bacillus form an
operon (qcrABC) similar to the proteobacterial fbcABC operon, but comparison of the corresponding aa sequences of the
FeS protein and cytochrome b indicate that Bst sequences are most similar to cyanobacterial counterparts.
The
thermophilic Bacillus cytochrome c is
quite unique; it consists of a subunit IV-like N terminus with three
trans-membrane segments, and a hydrophilic cytochrome c-like C
terminus, unlike either proteobacterial cytochrome c
or cyanobacterial cytochrome f. A very similar gene, qcrABC, having the same operon structure and close sequence
homologies, was very recently found in B. subtilis in the
course of studies on
F-directed transcription during
sporulation(33) . Although the B. subtilis cytochrome
reductase, has not been purified, the gene structure suggests the
presence of cytochrome reductase very similar to that of the
thermophilic bacilli (cytochrome b
c
complex). A recent report on the green sulfur bacterium C.
limicola showed petCB for cyanobacterial type-FeS protein
and cytochrome b. The latter is not split but is more similar
to the cyanobacterial cytochrome b
plus subunit IV gene than proteobacterial fbcB gene(23) . The gene for cytochrome c
is missing in this operon of C. limicola. Gene structure
of cytochrome reductases of eubacteria described above suggest the
following evolutionary scenario. The commonote had a long gene for
cytochrome b, while the original genes for the FeS protein and c-type cytochrome occurred at separate places. Then,
proteobacteria integrated the genes for their FeS protein and
cytochrome c
into operon structure, while the
green sulfur bacteria recombined with a slightly different FeS gene
(note that its N-terminal part is cyano-bacterial type). Gene
separation, introducing cytochrome b
and subunit
IV, due to formation of a stop codon occurred next in a cyanobacterial
ancestor. The ancestor of Bacillus recombined the gene for
cyanobacterial-type FeS protein upstream of the gene for cytochrome b
in addition to obtaining the gene for Bacillus small cytochrome c at the 3`-end of the gene
for subunit IV in order to donate electrons to cytochrome c.
Although proteobacterial cytochrome c
, Bacillus cytochrome c
, and cyanobacterial
cytochrome f are similar in size, their origins seem to be
quite different. It is interesting to find that cytochrome bc
/b
f complexes,
common for photosynthesis, are composed of different subunits formed
through protein domain swapping. In the terminal oxidase superfamily,
only subunit I bearing low spin heme and high spin heme-copper
binuclear center is completely conserved as the catalytic unit, while
the presence and origin of other subunits is
variable(34, 35) .
The nucleotide sequence(s) reported in this paper has been submitted to the GenBank(TM)/EMBL Data Bank with accession number(s) D83789[GenBank].