Cytochrome c
H Tuppy and G Kreil, University of Vienna, Vienna, Austria
ã 2013 Elsevier Inc. All rights reserved.
This article is reproduced from the previous edition, volume 1, pp. 535–538, ã 2004, Elsevier Inc.
Glossary
Apoptosis A means for the multicellular organism to
eliminate cells that are no longer needed or are damaged,
also called ‘programmed cell death’.
Cytochromes Literally cell pigments, heme
proteins chiefly involved in cell respiration and
energy supply.
Heme An iron porphyrin complex that is attached
(as a prosthetic group) to protein moieties in the red
Cytochrome c is a heme protein that is present in and can easily
be isolated from mitochondria of all eukaryotic organisms. The
amino acid sequence of the protein moiety was among the first
sequences that could be elucidated. This was the starting point
for comparative studies about sequence variations found in
cytochrome c from a wide range of species. A phylogenetic
tree constructed on the basis of this information was found
to be biologically significant and became exemplary for
subsequent studies on molecular evolution. The function of
cytochrome c in the respiratory chain as an electron carrier is
well established. More recently, an additional role of cyto-
chrome c was discovered: its release from mitochondria into
the cytosol triggers apoptosis – the programmed cell death.
Introduction
Cytochromes are proteins that contain heme as their prosthetic
group and whose principal biological function, in the cells of
animals, plants, and microorganisms, is electron transport. The
foundations of the knowledge of heme proteins and their roles
as electron carriers in cell respiration were laid by David Keilin
(1887–1963),
80 years ago. In the cytochromes, the iron
which is coordinately linked with four nitrogen atoms within
the prosthetic group and with two additional ligands provided
by the protein moiety, can alternate between a reduced Fe2þ
and an oxidized Fe3þ state. Different classes of cytochromes
(a type, b type, and c type) differ in the nature of their heme
prosthetic groups. They can be observed and differentiated
spectroscopically, on the basis of characteristic absorption
bands. The absorption peaks of reduced cytochrome c and
other c-type cytochromes (such as c1) are at
550, 520, 416,
and 270 nm. The b-type and a-type cytochromes absorb visible
light at higher wavelengths. In eukaryotic cells, cytochromes of
types a, b, and c are found predominantly within mitochondria.
In the mitochondrial respiratory chain, cytochrome c accepts
electrons from complex III, which contains cytochromes b
and c1 (bc1), and transmits them to complex IV (cytochrome
oxidase), which has two heme prosthetic groups (aa3).
These respiratory complexes, with their respective cyto-
chromes, are firmly integrated in the inner membrane of the
blood pigment hemoglobin, in the cytochromes and in
many other heme proteins.
Mitochondria Intracellular organelles, the main site of
oxidative metabolism that supplies the cell with utilizable
energy in the form of adenosine triphosphate. In the course
of cellular respiration, elementary oxygen is taken up by
mitochondria and reduced to water. The electrons required
for reduction are transmitted to oxygen via the respiratory
chain, one of the electron transporters being cytochrome c.
mitochondrion. Cytochrome c, by contrast, is only loosely
bound in the space between the inner and outer mitochondrial
membranes and shuttles between bc1 and aa3. As shown by
Keilin and others, cytochrome c can be easily removed experi-
mentally from and reincorporated into isolated mitochondria,
their respiratory capacity thereby being impaired and restored,
respectively. It has recently been discovered that the release of
cytochrome c from mitochondria into the cytoplasm is an
important step in programmed cell death. This participation
in apoptosis of eukaryotic cells is now considered to be another
significant function of cytochrome c.
Cytochrome c and c-type cytochromes occur in all eukary-
otic organisms. The comparative analysis of the protein struc-
tures of cytochromes isolated from a multitude of different
species of organisms has made it possible to establish evolution-
ary relationships between them. The phylogenetic tree based on
the slow changes of the structure of cytochrome c in the course
of evolution is most impressive and biologically relevant.
The c-type cytochromes are also present in prokaryotes,
where they may function in a respiratory chain or in photosyn-
thesis; examples are cytochrome c2 from Rhodospirillum rubrum
(purple bacteria) and cytochrome c551 from Pseudomonas aero-
ginosa (Gram-negative bacteria). These are distantly related to
mitochondrial cytochromes c; however, a more detailed dis-
cussion of these diverse hemoproteins is outside the scope of
this article.
Attachment of the Heme Prosthetic Group to
Cytochrome c Apoprotein
The prosthetic group of c-type cytochromes, such as cyto-
chrome c and c1 in mitochondria and cytochrome f in chlor-
oplasts, unlike that of a- and of b-type cytochromes, is
covalently linked to the polypeptide moiety. The thiol groups
of two cysteinyl residues in a Cys–X–Y–Cys–His peptide motif
are attached to two vinyl groups of heme through thioether
bonds. This linkage is resistant to heat and hydrolysis, but can
be broken with the help of silver or mercury salts. Thus, the
polypeptide moiety of cytochrome c, or of heme peptides
derived from it by proteolytic cleavage, could be obtained. In
599
600 Bioenergetics | Cytochrome c
eukaryotic cells, the apoprotein of cytochrome c is encoded by a
nuclear gene, translated on cytoplasmic ribosomes, and trans-
located into the intermembrane space of mitochondria, where
an enzyme, cytochrome c heme lyase, combines it with heme.
An in vitro synthesis of a microbial c-type cytochrome from its
apoprotein and reduced heme has recently been achieved.
Amino Acid Sequence Studies
Cytochrome c is a small, water-soluble protein that can easily
be isolated and purified from cells and tissues of many differ-
ent eukaryotic organisms. In proteolytic digests of cytochrome c,
one fragment containing the covalently linked heme group
can be separated from all the other ones. Studies on the
amino acid sequence of these heme peptides isolated from
cytochromes c of diverse origin thus became feasible. It turned
out that certain sequence elements, as the one mentioned
in the Introduction, were highly conserved from yeast to
mammalian cytochromes c.
Expanding on this early work, cytochrome c from horse
heart was one of the first proteins whose complete amino
acid sequence could be determined. It consists of a single
polypeptide chain of only 104 residues that could be analyzed
even with, by modern standards, rather primitive tools avail-
able around 1960. The protein contains many (up to 19) lysine
residues, whose positive charges can form ionic bonds with
constituents of the inner mitochondrial membrane.
With the horse sequence as a reference, cytochrome c isolated
from diverse species was subsequently analyzed in quick succes-
sion. These included other mammals (human, rhesus monkey,
pig, dog, rabbit, whale, and kangaroo), birds (chicken, pigeon,
king penguin, and turtle), reptiles (rattlesnake and alligator),
amphibia (bullfrog), fish (tuna, carp, and dogfish), and the
chordate pacific lamprey. Further, with unabated pace, addi-
tional sequences were determined for cytochromes c from flies,
moths, yeasts, Neurospora, and a large variety of plants. By now,
we know the sequence of this protein from more than 100
eukaryotic species. As a result of these efforts, cytochrome c
became the first and still is a standard example for studies on
the molecular evolution of proteins. Nuclear genes encoding
cytochrome c have been analyzed from various species. The
mammalian gene contains a small intron in the coding sequence
and a larger one upstream of the initiation codon.
The recent progress in the sequencing of whole eukaryotic
genomes has also yielded information about the number of
cytochrome c genes in various species. For example, in the
human genome, a single gene for cytochrome c is present on
chromosome 7, along with 49 pseudogenes located on many
different chromosomes. These are mostly processed pseudo-
genes, which were formed by retro-transcription of a messen-
ger RNA. In rodents, two cytochrome c genes are expressed, one
of them exclusively in the testis. This latter gene is still present
as a pseudogene in the human genome.
Evolutionary Aspects
Cytochrome c is a highly conserved protein, which retained
many structural characteristics over the eons of evolution of
animals and plants. This makes it an ideal tool for amino acid
sequence comparisons over a wide range of species (Figure 1).
From the sequence differences, a molecular pedigree can be
constructed based on the original assumption, now corrobo-
rated by many examples, that the longer ago the common ances-
tor of two species existed, the more amino acid changes will have
accumulated since. As it turned out, the phylogenetic trees con-
structed on the basis of the sequence of a single protein were
surprisingly similar to those derived from comparative anatomy,
fossils, etc. Moreover, as cytochrome c could be extracted from
species for which no reliable data were available from other
sources, these could now also be included in this analysis.
In this comparison of cytochromes c from diverse eukary-
otic species, it was found that more than a quarter of the amino
acids have been conserved during more than a billion years of
evolution. Among these immutable residues are the two
cysteines to which the heme is bound via thioether linkages.
The central iron of the planar heme is bound not only to four
nitrogen atoms of the porphyrin but also to the side chains of
two invariant amino acids of the protein, one being a histidine
adjacent to the second cysteine (residue #18 in the vertebrate
sequences), the other the sulfur atom of methionine 80, pres-
ent on opposite sides. Moreover, certain amino acids with
hydrophobic side chains, some of which line the crevice
where the heme is buried, are also conserved. Surprisingly, it
was found that several glycine residues in the sequence were
also invariant. Glycine is the smallest amino acid containing
only a hydrogen atom where the 19 others have more or less
0.1
Human / chimpanzee
Mouse
Rabbit
Horse
Pigeon
Chicken
King penguin
Turtle
Bullfrog
Tuna
Carp
Lamprey
Starfish
Prawn
Fruit fly
Wheat
Sunflower
Mung bean
Ginkgo
Fission yeast
Neurospora
Baker’s yeast
Figure 1 Phylogenetic tree constructed from the amino acid
sequences of cytochrome c using the ClustalW program. The length of
the branches corresponds to evolutionary distances (0.1 ¼ 10%
amino acid substitutions).

bulky side chains. From the three-dimensional structure, this
first puzzling observation could be explained. At these sites,
there is simply no space in this tightly folded protein and every
other amino acid would presumably prevent proper folding of
the polypeptide chain and thus be deleterious. In addition,
some charged amino acids, mostly lysines, have been con-
served. These may be essential for the ionic interaction with
two of its reaction partners, as well as with acidic phospholi-
pids present in the inner mitochondrial membrane.
Three-Dimensional Structure
Using X-ray crystallographic analysis, the three-dimensional
structure of tuna cytochrome c in its oxidized and reduced
forms could be determined. As shown by these studies, the
polypeptide chain is tightly wrapped around the heme, which
sits in a deep pocket, exposed only on one edge to the solvent. In
accordance with the oil-droplet model of folded proteins, the
hydrophobic amino acids are mostly buried inside the protein,
some of them surrounding the heme. In the respiratory chain
located in the inner membrane of mitochondria, cytochrome c
shuttles between a reduced ferro- (Fe2þ) and ferri- (Fe3þ) form.
In this redox cycle, the protein changes its conformation, with
the reduced form having a more compact structure.
Interactions of Cytochrome c in the Respiratory Chain
Cytochrome c is loosely bound to the outer surface of the
inner mitochondrial membrane. In the respiratory chain, an elec-
tron is transferred to cytochrome c from cytochrome bc1. These
cytochromes are part of a large, membrane-bound complex, also
termed complex III composed of 11 proteins. Subsequently, the
electron is delivered to the cytochrome aa3 of the complex IV, the
cytochrome oxidase, which contains 13 proteins. Some of the
molecular details of this electron transport have recently also
been clarified. In the crystal structure of the yeast cytochrome bc1
complex with its bound substrate cytochrome c, the heme groups
of cytochrome c1 and c are in close proximity (see Figure 2). This
suggests that the redox process takes place by direct heme-to-heme
electron transfer. The same may also be true for the oxidation of
cytochrome c by cytochrome aa3.
In both redox reactions, a proton is pumped across the
inner membrane. The proton gradient thus formed drives
the adenosine triphosphate (ATP) synthetase motor, which
results in the synthesis of ATP.
Cytochrome c and Apoptosis
Cells that are damaged or are no longer needed may undergo a
form of cell death that is controlled and executed by intracel-
lular programs. One major apoptotic program involves the
release of cytochrome c from the intermembrane space of
mitochondria into the cytosol, together with some other mito-
chondrial proteins. Once in the cytoplasm, cytochrome c com-
bines with an apoptosis activating factor-1 (Apaf-1) and thus
triggers the assembly of a multimeric protein complex, the
so-called apoptosome. An inactive proform of a proteolytic
enzyme, procaspase-9, is then recruited to the apoptosome
and activated. Active caspase-9, in turn, activates other
Bioenergetics | Cytochrome c 601
Figure 2 Part of the yeast cytochrome bc1 complex and the bound
substrate cytochrome c (Protein Data Bank, accession number 1KYO).
Cytochrome c1, yellow; cytochrome c, magenta; hemes, red.
caspases, whose proteolytic actions finally lead to cell death.
The crucial role of cytochrome c in this process is shown by the
fact that microinjection of cytochrome c into the cytoplasm
suffices to induce apoptosis in several cell lines. In the interac-
tion with Apaf-1, as in the interaction with cytochrome c1, the
exposed heme edge of cytochrome c is involved.
When cytochrome c molecules are released from the
mitochondrion, they have to pass through pores in the outer
membrane of the organelle. A group of related proteins, the
Bcl-2 family, regulates apoptosis by controlling the permeability
of this membrane. Bcl-2 itself and other antiapoptotic proteins
are located in the outer mitochondrial membrane and inhibit
cytochrome c release. Conversely, release of cytochrome c is
triggered by proapoptotic members of the Bcl-2 family, which
are induced by a number of stimuli to translocate to the mito-
chondria. Since one of the functions of apoptosis is to help the
multicellular organism to eliminate cancer cells, the pro- and
antiapoptotic Bcl-2 family members are considered to be onco-
genic and antioncogenic proteins, respectively. They control
whether cytochrome c, besides being a vital mediator of elec-
tron transfer in respiration, also gets involved in cell death.
See also: Bioenergetics: Cell Death by Apoptosis and Necrosis;
Cytochrome Oxidases, Bacterial; Heme Synthesis; Purple Bacteria:
Electron Acceptors and Donors; Purple Bacteria: Photosynthetic
Reaction Centers; Protein/Enzyme Structure Function and
Degradation: Heme Proteins.
Further Reading
Dickerson RE (1972) The structure and history of an ancient protein. Scientific American
226: 58–70.
Dickerson RE (1980) Cytochrome c and the evolution of energy metabolism. Scientific
American 242: 98–109.
Hengartner MO (2000) The biochemistry of apoptosis. Nature 407: 770–776.
Lange C and Hunte C (2002) Crystal structure of the yeast cytochrome bc1 complex
with its bound substrate cytochrome c. Proceedings of the National Academy of
Sciences of the United States of America 99: 2800–2805.
Saraste M (1999) Oxidative phosphorylation at the fin de siècle. Science 283: 1488–1493.
Slater EC (2003) Keilin, cytochromes, and the respiratory chain. Journal of Biological
Chemistry 278: 16455–16461.
Zhang Z and Gerstein M (2003) The human genome has 49 cytochrome c pseudogenes,
including a relic of a primordial gene that still functions in mice. Gene 312: 61–72.