Heme/Copper Terminal Oxidases
Shelagh Ferguson-Miller*
Department of Biochemistry, Michigan State University, East Lansing, Michigan 48824
Gerald T. Babcock*
Department of Chemistry, Michigan State University, East Lansing, Michigan 48824
Received August 6, 1996 (Revised Manuscript Received September 2, 1996)
Contents
A. Introduction 2889
B. Insights from the Atomic Resolution Structures of
Cytochrome c Oxidases
2890
1. Electron-Transfer Pathways 2891
2. Proton Exit Routes 2892
3. Electron Entry Sites 2893
4. Coupling Mechanisms and Proton Transfer
Pathways
2894
5. Water and Oxygen Channels 2894
6. Unique Aspects of the Mammalian Oxidase
Structure
2895
C. Bioactivation and Reduction of Dioxygen 2895
1. Oxygen Toxicity and Electron-Transfer
Control
2895
2. Proton-Transfer Control and the Proton Pump
in Heme/Copper Terminal Oxidases
2896
D. Reaction of Cytochrome Oxidase with
Oxygen-Containing Substrates
2898
1. Dioxygen Binding and the Initial Oxy
Intermediate
2898
2. The Decay of the Oxy Species and the
Peroxide Issue
2899
3. The Peroxy and Ferryl Levels in the
Reduced-Enzyme/O
2
Reaction
2900
4. The Peroxy and Ferryl Levels in the
Mixed-Valence Enzyme/O
2
and
Oxidized-Enzyme/H
2
O
2
Reactions
2903
E. Proton Control, Proton-Coupled Electron
Transfer, and Proton Pumps
2904
F. Conclusions 2905
A. Introduction
Spatially well-organized electron-transfer reactions
in a series of membrane-bound redox proteins form
the basis for energy conservation in both photosyn-
thesis and respiration. The membrane-bound nature
of the electron-transfer processes is critical, as the
free energy made available in exergonic redox chem-
istry is used to generate transmembrane proton
concentration and electrostatic potential gradients.
These gradients are subsequently used to drive ATP
formation, which provides the immediate energy
source for constructive cellular processes.
The terminal heme/copper oxidases in respiratory
electron-transfer chains illustrate a number of the
thermodynamic and structural principles that have
driven the development of respiration (see refs 1-6,
for recent reviews). This class of enzyme reduces
dioxygen to water, thus clearing the respiratory
system of low-energy electrons so that sustained
electron transfer and free-energy transduction can
occur. By using dioxygen as the oxidizing substrate,
free-energy production per electron through the chain
is substantial, owing to the high reduction potential
of O
2
(0.815 V at pH 7). For example, if the initial
electron donor is NADH, more than 1.1 eV/electron
through the respiratory chain is available for ATP
synthesis. Moreover, the dioxygen binding and ac-
tivating site in the terminal oxidases, which com-
prises the binuclear heme a
3
/Cu
B
center (section B),
is ideally suited to using O
2
as an oxidizing substrate.
Very little leakage of potentially damaging, partially
reduced oxygen species (O
2
•-
,O
2
2-
,OH
•
) occurs from
this site; recent work implicates the quinone cofactors
as the principal loci for the generation of reactive
oxygen species in the respiratory process.
7
The high-spin heme/Cu
B
site is also constructed so
as to operate dioxygen reduction at low overpotential.
As opposed to most electrochemical methods for
dioxygen reduction, where overpotentials greater
than 1 V are commonly encountered, the dioxygen
overpotential in the terminal oxidases is less than
0.3 V. This feature of the binuclear center is critical
in maximizing respiratory free-energy production.
Firstly, it allows the use of fairly high potential
reductants as its electron source: in the cytochrome
c oxidases, the potential of the cytochrome c donor is
250 mV; in the quinol terminal oxidases, the quinol
potential is only ∼200 mV more negative. The net
result of these energetics is that the driving force
available for redox-linked proton translocation in
regions of the respiratory chain prior to the terminal
oxidase is maximized. Secondly, the low overpoten-
tial associated with O
2
reduction, in concert with the
vectorial nature of electron transfer through the
enzyme, allows the terminal oxidases themselves to
contribute directly to the transmembrane free-energy
gradient, that is, they operate as redox-linked proton
pumps.
8
The proton-translocating activity of the
enzyme occurs with a stoichiometry that approaches
one proton pumped per electron delivered to dioxy-
gen. Thus, we can write the overall reaction cata-
lyzed by the terminal oxidases as
where D
red
and D
ox
represent the reduced and oxi-
dized forms of the immediate electron donors to the
4D
red
+ O
2
+ 8H
+
in
f 4D
ox
+ 2H
2
O + 4H
+
out
2889Chem. Rev. 1996, 96, 2889−2907
S0009-2665(95)00051-3 CCC: $25 00 1996 American Chemical Society
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enzyme and H
+
in
and H
+
out
indicate that protons are
taken up and released vectorially with respect to the
respiratory membrane.
The past 4 years have seen rapid developments in
our understanding of the inner working of this
remarkable class of enzyme. The enzyme from two
sources has been crystallized and its structure de-
termined to high resolution. In fact, the local struc-
ture about the metal centers in subunit I determined
by mutagenesis/spectroscopic approaches predated
that of the crystal structures and correctly predicted
the amino acid ligands to the redox cofactors in the
resting form of the enzyme. Concomitant with the
structural and mutagenetic progress has been the
rapid development of time-resolved spectroscopic
techniques that have provided deep insight into
mechanistic aspects of the functional events catalyzed
by the enzyme. Although progress in understanding
the operation of the enzyme has been substantial,
there are a number of areas, particularly those
relating to the interplay between proton and electron
currents, in which our ability to link observed phe-
nomena to structure and activity of the protein is only
rudimentary. In this review, we will summarize the
progress that has been made, identify areas in which
consensus is emerging, and, finally, indicate those
aspects of enzyme function that remain problematic.
B. Insights from the Atomic Resolution
Structures of Cytochrome c Oxidases
The commonality of the electron-transfer systems
in eukaryotes and prokaryotes was observed by
Keilin in 1925,
9
but only in the past 10 years have
investigators exploited this fact, using the structur-
ally simpler, genetically accessible bacterial systems
to study the mechanism of energy transduction. In
the case of cytochrome oxidase, this approach has led
to major advances in our understanding of structure
and mechanism, through the use of mutagenesis
combined with spectroscopy and ultimately crystal-
lography, culminating in the recent, successful solu-
tion of a 2.8 Å resolution X-ray structure of a
bacterial cytochrome c oxidase.
10
This amazing feat
was matched by the simultaneous achievement of an
atomic-level resolution structure of the 13 subunit
mammalian cytochrome c oxidase.
11,12
Coincident
with these landmarks in membrane protein crystal-
lography was the determination of a 2.5 Å structure
of a soluble domain of oxidase containing an engi-
neered Cu
A
center.
13
This sudden wealth of structural information has
had, and will have, an enormous impact on the field.
Although analysis of site-directed mutants success-
fully predicted many aspects of the active site struc-
ture, of the positions of metal centers in the pro-
tein,
14,15
and of the key residues in likely proton
channels,
16-18
confirmation and extension of these
pieces of knowledge by a complete structure provides
the basis for far more powerful analysis of mecha-
nism.
Shelagh Ferguson-Miller is Professor and Associate Chair of Biochemistry
at Michigan State University. Born in Toronto, Canada, she did
undergraduate studies at the University of Toronto in Physiology and
Biochemistry. She received a Master’s degree in Biochemistry from the
University of Toronto in 1966 and a Ph.D. in Biochemistry in 1971 with
Henry Lardy at the Enzyme Institute, University of Wisconsin, Madison.
After postdoctoral studies with George Radda at Oxford University, U.K.,
and Emmanuel Margoliash at Northwestern University, Evanston, IL, she
was appointed Assistant Professor of Biochemistry at Michigan State
University in 1978. Current research interest is in the area of bioener-
getics: electron and proton translocation in cytochrome c oxidase,
cytochrome c docking with cytochrome oxidase, and design and analysis
of detergents for crystallizing membrane proteins. Publications include
over 75 papers in refereed journals. She is on the editorial boards of
Biochimica et Biophysica Acta and the Journal of Bioenergetics and
Biomembranes and is Chairperson of the Gordon Conference on
Bioenergetics in 1997. She has recently received the Distinguished Faculty
Award from Michigan State University.
Gerald T. Babcock was born in Minneapolis, MN. He did his
undergraduate work at Creighton University in Omaha, NB, where an
interest in chemistry developed that was sparked in high school.
Professors Zebolsky, Snipp, and Takamura were instrumental in focusing
this interest toward the exploration of biological phenomena with physical
chemical approaches. At the University of California, Berkeley, he did
his graduate work under the guidance of Professor Ken Sauer and became
fascinated with photosynthetic processes, particularly the photochemical
and chemical reactions that produce O
2
from water. He stayed in Berkeley
for an additional year after receiving his Ph.D. in 1973 and then moved,
as an NIH postdoctoral fellow, to Professor Graham Palmer’s lab at Rice
University, where he studied the other half of the water/oxygen cycle in
Nature, i.e., the reduction of O
2
to water by cytochrome oxidase. In 1976,
he moved to Michigan State University, where he is now Professor and
Chairman in the Department of Chemistry. He continues his interests in
the biological aspects of water/oxygen chemistry and uses spectroscopic
techniques, primarily Raman and electron magnetic resonance, to obtain
deeper insights into the metabolism of this redox couple in a variety of
enzymes. At present, he is an Associate Editor of the Annual Reviews
of Physical Chemistry and a member of the Editorial Advisory Board for
Biochemistry. He was a visiting professor at the College de France and
a Phillips Lecturer at Haverford College. His interests outside science
include bicycling, squash, and white-water canoeing, the latter of which
he shares on a regular basis with members of his research group.
2890 Chemical Reviews, 1996, Vol. 96, No. 7 Ferguson-Miller and Babcock
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