REVIEW ARTICLE
Cytochrome
c
from a thermophilic bacterium has provided insights
into the mechanisms of protein maturation, folding, and stability
Yoshihiro Sambongi
1
, Susumu Uchiyama
2,
*, Yuji Kobayashi
2
, Yasuo Igarashi
3
and Jun Hasegawa
4
1
Graduate School of Biosphere Sciences, Hiroshima University, Japan;
2
Faculty of Pharmaceutical Science, Osaka University, Japan;
3
Department of Biotechnology, University of Tokyo, Japan;
4
Daiichi Pharmaceutical Co., Ltd, Tokyo, Japan
Cytochrome c is widely distributed in bacterial species, from
mesophiles to thermophiles, and is one of the best-charac-
terized redox proteins in terms of biogenesis, folding, struc-
ture, function, and evolution. Experimental molecular
biology techniques (gene cloning and expression) have
become applicable to cytochrome c, enabling its engineering
and manipulation. Heterologous expression systems for
cytochromes c in bacteria, for use in mutagenesis studies,

Cys-His motif [1–4]. Site-directed mutagenesis studies of
cytochrome c are at a relatively early stage, because of the
difficulty of expressing the holoprotein heterologously.
However, during the last decade, it has been found that
bacteria have specific cellular apparatus for covalent
attachment of a heme to the cytochrome c polypeptide
[1–4]. Knowledge obtainedfrom studies on the cytochrome c
biogenesis pathway in bacteria has been used to produce
heterologous cytochromes c in large quantities, which has
facilitated mutagenesis studies and structure analysis.
In this review, we will summarize how bacterial cyto-
chromes c have been used in recent mutagenesis and
structure studies to elucidate protein stability. Through such
investigations, we have gained insight into the molecular
mechanisms underlying cytochrome c maturation and
folding as well as stability. Bacterial cytochrome c has
contributed greatly to our understanding in these areas, and
is one of the most successful model proteins. The basic ideas
obtained with cytochromes c should be applicable to other
proteins of industrial and/or medical interest.
CYTOCHROME c AS A MODEL FOR
PROTEIN STABILITY STUDIES
Proteins isolated from thermophilic bacteria are usually
stable to heat and chemical denaturants, indicating that they
must have most of the determinants of protein stability.
Initial clues to the relationship between protein structure
and stability can be obtained by pairwise sequence com-
parison of homologous proteins from thermophiles and
mesophiles. The cytochromes c, which play a central role in
electron-transport chains in both thermophilic and

to the polypeptide chain [5]. The amino-acid sequence
(Fig. 1) and main chain folding (Fig. 2) of HT c-552 from
this thermophile closely resemble those of the 82-amino-acid
cytochrome c-551 from a mesophile, Pseudomonas aerugi-
nosa (PA c-551). Comparisons of the two proteins indicated
that the amino-acid residues are 56% identical [5], and that
the root mean square deviation for their main chain folding is
within 1 A
˚
[6]. However, as expected from the difference in
their optimal growth temperatures (H. thermophilus, 70 °C;
P. aeruginosa, 37 °C), HT c-552 is more stable to heat and
chemical denaturants than PA c-551 [7–9]. For instance, the
former has a significantly higher mid-point denaturation
temperature than the latter, as judged both spectrophoto-
metrically and calorimetrically. As described below, inves-
tigation of the relationship between the 3D structures and
thermodynamic properties accompanying protein denatu-
ration of HT c-552 and PA c-551 (wild-types and mutants)
has revealed the molecular mechanism underlying protein
stability. The difference in stability between them can be
attributed to differences in side-chain interactions in a few
select regions [9] (see below).
Other homologous cytochromes
c
To determine the amino-acid residues responsible for
protein stability, it would be better if sequence information
was available from a larger number of homologous proteins
in a variety of bacteria that differ in optimal growth
temperature. Many homologous cytochromes c that exhibit

sequences will provide valuable information on the mech-
anism underlying protein stability.
BIOGENESIS OF BACTERIAL
CYTOCHROMES c
To confirm experimentally the amino-acid residues respon-
sible for the stability indicated by sequence and 3D structure
comparisons, site-directed mutagenesis needs to be per-
formed on a series of homologous cytochromes c.Forthis,
the cytochrome c gene needs to be heterologously expressed
as a holoprotein that has a covalently attached heme and is
in a correctly folded form, like the authentic proteins
isolated from the original bacteria. Heme attachment, which
must occur regardless of whether the cytochrome c gene is
Fig. 1. Amino-acid sequence comparison of cytochromes c homologous
to HT c-552. Amino-acid sequences are aligned using residue numbers
(every 10) of PA c-551. HT, Hydrogenobacter thermophilus;PA,
Pseudomonas aeruginosa;PS,Pseudomonas stutzeri;PZ,Pseudomonas
stutzeri Zobell; PM, Pseudomonas mendocina;PD,Pseudomonas
denitrificans;PF,Pseudomonas fluorescens;AV,Azotobacter vinelandii;
NE, Nitrosomonas europaea; AA, Aquifex aeolicus.
Fig. 2. 3D structural comparison of HT c-552 and PA c-551. The
main-chain folding of HT c-552(red)isoverlaidwiththatofPAc-551
(green).
3356 Y. Sambongi et al.(Eur. J. Biochem. 269) Ó FEBS 2002
endogenous or exogenous, is a unique step during cyto-
chrome c biogenesis. To be able to attempt heterologous
expression of the cytochrome c gene, it is important to
determine how the heme attaches to the polypeptide in the
cell and how its functional structure is formed. The path-
way of cytochrome c biogenesis has been extensively studied

A defect in an integral membrane protein, DsbD (also
known as DipZ), was first characterized as an E. coli
mutation that prevented the synthesis of mature cyto-
chrome c in the periplasm [17]. DsbD contains a domain
with potential disulfide isomerase activity facing the
periplasm [18–20]. Other Dsb proteins, DsbA and DsbB,
which have been determined to oxidize cysteine thiols to
form the internal disulfide bonds of many proteins in the
E. coli periplasm, are also required for cytochrome c
biogenesis [21,22].
Cytochrome c biogenesis in dsbD mutant cells can be
restored by adding low-molecular-mass thiol compounds to
the growth medium [17], and that in dsbA and dsbB mutants
by adding disulfide compounds [22]. These complementa-
tion results are consistent with the general role of Dsb
proteins in the regulation of the thiol–disulfide redox
conditions during periplasmic protein folding. Although
no biochemical evidence for the requirement of the Dsb
system during cytochrome c biogenesis has been obtained
yet, the genetic evidence suggests that thiol–disulfide redox
control is also essential for cytochrome c biogenesis in the
periplasm. Importantly, these results indicated that the level
of cytochrome c production could easily be controlled by
the thiol–disulfide redox potential, and this is the case, as
described below.
EXPRESSION OF EXOGENOUS
CYTOCHROME c GENES IN BACTERIA
For mutagenesis studies and structure analysis, it is
necessary to obtain heterologously expressed, mature holo-
cytochromes c in large quantities. Knowledge obtained

be used for further biochemical analysis as if we are dealing
with the Ônative proteinsÕ.
Control of production level
The yields of heterologously expressed cytochromes c may
depend on the copy numbers of the plasmids used. In
addition, coexpression with ccm genes is effective for higher
levels of plasmid-borne cytochrome c gene expression [27].
Not only the protein factors functioning in the periplasm,
but also low-molecular-mass thiol/disulfide compounds,
which can maintain the periplasmic redox balance [28],
successfully control the cytochrome c production level. For
instance, the yields of exogenous and endogenous
cytochromes c reach about 10% of the total periplasmic
protein fraction in E. coli with the addition of disulfide
compounds to the medium [22]. Furthermore, a certain
E. coli strain, JCB7120, can produce exogenous holo-
(PA c-551) up to 30% of the total periplasmic protein level
[8], although the mechanism underlying this high expression
in this strain is not yet known. Now, using bacterial
expression systems, we can obtain large amounts of
holocytochromes c, which in terms of visible absorption
spectra and other properties are indistinguishable from the
native proteins. This progress has facilitated mutagenesis
Ó FEBS 2002 Cytochrome c structure and stability (Eur. J. Biochem. 269) 3357
and structure analyses of bacterial cytochromes c, including
HT c-552 and PA c-551.
MUTAGENESIS STUDIES FOR
STABILITY
In general, site-directed mutagenesis is a powerful tool for
investigating the relationship between protein structure and

stability in an additive manner, and hoped that we would be
able to clearly determine the stabilizing factors by muta-
genesis studies.
Engineering stable proteins
To characterize the factors that affect protein stability, we
attempted to achieve maximal enhancement of the stability
of PA c-551 by introducing minimal mutations into spa-
tially separate regions. Five amino-acid residues in
PA c-551, which were selected on structure comparison,
were substituted with those found at the corresponding
positions in HT c-552, and the stabilities of the resulting
PA c-551 mutants were measured. A single mutation [Val78
to Ile (V78I)] and double mutations [Phe7 to Ala/Val13 to
Met (F7A/V13M) and Phe34 to Tyr/Glu43 to Tyr (F34Y/
E43Y)] in the three regions of PA c-551 each individually
enhanced the overall protein stability [8]. These studies,
together with structure analysis, provided substantial clues
to the mechanism of protein stability in HT c-552. Ala7/
Met13 and Ile78 fill small spaces present in the correspond-
ing regions of PA c-551, and Tyr34/Tyr43 cause a favorable
electrostatic interaction. These side-chain interactions may
contribute to the enhanced stability of HT c-552. It would
be worth trying to mutate HT c-552 so that it has the amino-
acid residues found in PA c-551, and to examine whether
the resulting HT c-552 mutants have decreased stability.
Surprisingly, a PA c-551 variant simultaneously carrying
the five mutations in the three separate regions (F7A/
V13M/F34Y/E43Y/V78I, quintuple mutant, Fig. 3) exhib-
its almost the same stability as that of natural HT c-552
(Fig. 4) [9]. This demonstrates that it is possible to convert a

hydrogen bonds between polar residues [30–32]. However,
these interactions are often related to each other in a protein
molecule, and subtle changes in them can affect the overall
stability. Thus, it is usually difficult to identify the exact
factors that contribute to the enhanced stability of the
proteins from thermophilic bacteria. This is reasonable
because proteins in the native state are stabilized by 10–
20 kJÆmol
)1
compared with those in the denatured state; the
energy is equivalent to the formation of only a few hydrogen
bonds. Therefore, to understand protein stability, it is
necessary to carry out precise comparative studies using
homologues exhibiting high sequence identity with similar
3D structures, but differing in stability. From such
comparisons, we must carefully detect subtle differences in
side-chain interactions, and examine their contributions to
the overall protein stability by mutagenesis studies. If the
interactions are spatially separated, their contributions may
be additive, in which case we can clearly identify protein-
stabilizing factors. The combination of precise comparison
of the structures of thermophilic and mesophilic homolog-
ous proteins and selection for multiple mutations in separate
regions is a valuable approach to elucidating the relation-
ship between structure and stability. This approach has been
successfully applied to cytochrome c (as discussed above),
triose phosphate isomerase [33], ribonuclease HI [34], and
cold shock protein [35].
Recent advances in genome projects have revealed the
gene resources of thermophilic bacteria, providing further

attachment and apoprotein folding through the follow-up
experiments.
Heterologous holo-(HT c-552), which has the heme
attached covalently, is found in the cytoplasm when the
truncated gene coding for the mature protein without the
original signal sequence is transformed into E. coli [23,36].
An apo-(HT c-552) variant carrying mutations at the heme
covalent binding site (C12A/C15A) has also been expressed
in the E. coli cytoplasm. This gene product was found to
have a compactly folded structure, which apparently differs
from that of the natural holoprotein with the heme attached
covalently [37]. The ÔfoldedÕ apoprotein aggregates into
amyloid fibrillar structures over a long time period [38], but,
in the presence of excess heme, it retains the prosthetic
group noncovalently like a b-type cytochrome [39]. In
contrast, mesophilic apocytochromes c seem to form a
random coil structure, and holoprotein formation does not
occur in the bacterial cytoplasm [23]. These observations
suggest that apo-(HT c-552) has a sufficiently ÔfoldedÕ
structure to incorporate a heme at moderate temperature,
possibly because of its thermostable properties. After
insertion of the heme, cytochrome c folding occurs. There-
fore, heme is not only required for the redox properties of
cytochrome c, but is also essential for correct protein
folding during cytochrome c biogenesis.
The unique case of cytoplasmic heme attachment also
leads to the hypothesis that covalent thioether bond
formation itself can proceed spontaneously without enzy-
matic assistance once the heme is inserted into the apopro-
tein [23,40]. Recently, a thermophile, Thermus thermophilus,

applicable to the elucidation of the dynamic features of
cytochromes c. It is of interest to characterize cytochrome c
with respect to protein maturation, folding, stability, and
function using a variety of combined experimental tech-
niques. Cytochrome c molecules will become very well
understood through such interdisciplinary methods.
ACKNOWLEDGEMENTS
We thank Ikuo Ueda for his support and encouragement, and Kazuaki
Nishio and Yuko Iko for critical reading of the manuscript.
REFERENCES
1. Ferguson, S.J. (2001) Keilin’s cytochromes: how bacteria use
them, vary them and make them. Biochem. Soc. Trans. 29, 629–
640.
2. Tho
¨
ny-Meyer, L. (2000) Haem–polypeptide interactions during
cytochrome c maturation. Biochim. Biophys. Acta 1459, 316–324.
3. Page, M.D., Sambongi, Y. & Ferguson, S.J. (1998) Contrasting
routes of c-type cytochrome assembly in mitochondria, chloro-
plast and bacteria. Trends Biochem. Sci. 23, 103–108.
4. Kranz, R., Lill, R., Goldman, B., Bonnard, G. & Merchant, S.
(1998) Molecular mechanisms of cytochrome c biogenesis: three
distinct systems. Mol. Microbiol. 29, 383–396.
5. Sanbongi, Y., Ishii, M., Igarashi, Y. & Kodama, T. (1989) Amino
acid sequence of cytochrome c-552 from a thermophilic hydrogen-
oxidizing bacterium, Hydrogenobacter thermophilus. J. Bacteriol.
171, 65–69.
6. Hasegawa, J., Yoshida, T., Yamazaki, T. & Sambongi, Y., Yu, Y.,
Igarashi, Y., Kodama, T., Yamazaki, K., Kyogoku, Y. &
Kobayashi, Y. (1998) Solution structure of thermostable cyto-

. Biochemistry 40, 13681–13689.
11. Dumont, M.E., Ernst, J.F., Hampsey, D.M. & Sherman, F. (1987)
Identification and sequence of the gene encoding cytochrome c
heme lyase in the yeast Saccharomyces cerevisiae. EMBO J. 6,
235–241.
12. Fabianek, R.A., Hennecke, H. & Tho
¨
ny-Meyer, L. (2000) Peri-
plasmic protein thiol:disulfide oxidoreductases of Escherichia coli.
FEMS Microbiol. Rev. 24, 303–316.
13. Mu
¨
hlenhoff, U. & Lill, R. (2000) Biogenesis of iron-sulfur proteins
in eukaryotes: a novel task of mitochondria that is inherited from
bacteria. Biochim. Biophys. Acta 1459, 370–382.
14. Schulz, H., Hennecke, H. & Tho
¨
ny-Meyer, L. (1998) Prototype of
a heme chaperone essential for cytochrome c maturation. Science
281, 1197–1200.
15. Ren, Q. & Tho
¨
ny-Meyer, L. (2001) Physical interaction of CcmC
with heme and the heme chaperone CcmE during cytochrome c
maturation. J. Biol. Chem. 276, 32591–32596.
16. Sambongi, Y., Stoll, R. & Ferguson, S.J. (1996) Alteration of
haem-attachment and signal-cleavage sites for Paracoccus
denitrificans cytochrome c
550
probes pathway of c-type cyto-

coccus denitrificans cytochrome c
550
requires targeting to the
periplasm whereas that of holo Hydrogenobacter thermophilus
cytochrome c
552
does not. Implications for c-type cytochrome
biogenesis. FEBS Lett. 340, 65–70.
24. Zhang, Y., Arai, H., Sambongi, Y., Igarashi, Y. & Kodama, T.
(1998) Heterologous expression of Hydrogenobacter thermophilus
cytochrome c-552 in the periplasm of Pseudomonas aeruginosa.
J. Ferment. Bioeng. 85, 346–349.
25. Ubbink, M., Van Beeumen, J. & Canters, G.W. (1992) Cyto-
chrome c
550
from Thiobacillus versutus: cloning, expression in
Escherichia coli, and purification of the heterologous holoprotein.
J. Bacteriol. 174, 3707–3714.
26. Fee, J.A., Chen, Y., Todaro, T.R., Bren, K.L., Patel, K.M., Hill,
M.G., Gomez-Moran, E., Loehr, T.M., Ai, J., Tho
¨
ny-Meyer, L.,
Williams, P.A., Stura, E., Sridhar, V. & McRee, D.E. (2000)
Integrity of Thermus thermophilus cytochrome c
552
synthesized by
Escherichia coli cells expressing the host-specific cytochrome c
maturation genes, ccmABCDEFGH: biochemical, spectral, and
structural characterization of the recombinant protein. Protein
Sci. 9, 2074–2084.

well as monomeric forms of the protein. Biochemistry 35, 4110–
4117.
34. Akasako, A., Haruki, M., Oobatake, M. & Kanaya, S. (1995)
High resistance of Escherichia coli ribonuclease HI variant with
quintuple thermostabilizing mutations to thermal denaturation,
acid denaturation, and proteolytic degradation. Biochemistry 34,
8115–8122.
35. Perl, D., Mu
¨
ller, U., Heinemann, U. & Schmid, F.X. (2000) Two
exposed amino acid residues confer thermostability on a cold
shock protein. Nat. Struct. Biol. 7, 380–383.
36. Sanbongi, Y., Yang, J.H., Igarashi, Y. & Kodama, T. (1991)
Cloning, nucleotide sequence and expression of the cytochrome
c-552 gene from Hydrogenobacter thermophilus. Eur. J. Biochem.
198, 7–12.
37. Wain, R., Pertinhez, T.A., Tomlinson, E.J., Hong, L., Dobson,
C.M., Ferguson, S.J. & Smith, L.J. (2001) The cytochrome c fold
can be attained from a compact apo state by occupancy of a
nascent heme binding site. J. Biol. Chem. 276, 45813–45817.
38. Pertinhez, T.A., Bouchard, M., Tomlinson, E.J., Wain, R.,
Ferguson, S.J., Dobson, C.M. & Smith, L.J. (2001) Amyloid fibril
formation by a helical cytochrome. FEBS Lett. 495, 184–186.
39. Tomlinson, E.J. & Ferguson, S.J. (2000) Conversion of a c type
cytochrome to a b type that spontaneously forms in vitro from apo
protein and heme: implications for c type cytochrome biogenesis
and folding. Proc.Natl.Acad.Sci.USA97, 5156–5160.
40. Sinha, N. & Ferguson, S.J. (1998) An Escherichia coli ccm (cyto-
chrome c maturation) deletion strain substantially expresses
Hydrogenobacter thermophilus cytochrome c