Active Site of Cytochrome
cbb3 |
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Authors: | Virve Rauham?ki Dmitry A Bloch Michael I Verkhovsky M?rten Wikstr?m |
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Institution: | Helsinki Bioenergetics Group, Program for Structural Biology and Biophysics, Institute of Biotechnology, University of Helsinki, P. O. Box 65 (Viikinkaari 1), 00014 Helsinki, Finland |
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Abstract: | Cytochrome cbb3 is the most distant member of the
heme-copper oxidase family still retaining the following major feature typical
of these enzymes: reduction of molecular oxygen to water coupled to proton
translocation across the membrane. The thermodynamic properties of the six
redox centers, five hemes and a copper ion, in cytochrome
cbb3 from Rhodobacter sphaeroides were studied
using optical and EPR spectroscopy. The low spin heme b in the
catalytic subunit was shown to have the highest midpoint redox potential
(Em,7 +418 mV), whereas the three hemes
c in the two other subunits titrated with apparent midpoint redox
potentials of +351, +320, and +234 mV. The active site high spin heme
b3 has a very low potential
(Em,7 -59 mV) as opposed to the copper center
(CuB), which has a high potential
(Em,7 +330 mV). The EPR spectrum of the ferric
heme b3 has rhombic symmetry. To explain the origins of
the rhombicity, the Glu-383 residue located on the proximal side of heme
b3 was mutated to aspartate and to glutamine. The latter
mutation caused a 10 nm blue shift in the optical reduced minus
oxidized heme b3 spectrum, and a dramatic change of the
EPR signal toward more axial symmetry, whereas mutation to aspartate had far
less severe consequences. These results strongly suggest that Glu-383 is
involved in hydrogen bonding to the proximal His-405 ligand of heme
b3, a unique interaction among heme-copper oxidases.The heme-copper oxidases form a family of enzymes that have structural
homology of the catalytic subunit in common
(1). This family of proteins,
characterized by six conserved histidine ligands of the redox cofactors,
ranges from classical, mitochondrial terminal oxidases to nitric-oxide
reductases, and the members have been classified according to evolutionary
relationships of their sequences
(2–4).
The bacterial cbb3-type cytochrome c oxidases
form a distinct, divergent subfamily within the heme-copper oxidases
(5). Terminal oxidases share
the catalytic activity of four-electron reduction of molecular oxygen to water
coupled to translocation of protons across the membrane
(6,
7). Cytochrome
cbb3, expressed in some bacteria as a sole terminal
oxidase, is characterized by its ability to maintain catalytic activity under
low oxygen tension (8), and it
has also been shown to have the capacity to translocate protons
(9).The Rhodobacter sphaeroides cytochrome cbb3 is
encoded by the ccoNOQP operon composed of four genes
(10). The catalytic subunit
CcoN homes a binuclear active site composed of a high spin heme
b3 and a nearby copper ion (CuB). There are
altogether four low spin hemes in the enzyme. In addition to a protoheme (heme
b) residing in the vicinity of the active site in subunit CcoN, there
are three hemes c present in the soluble domains of the two other
transmembrane subunits, a monoheme subunit CcoO and a diheme subunit CcoP
(11). There is yet one more
membrane-spanning subunit, CcoQ, without bound cofactors
(12). Although the catalytic
subunit shows homology to the other heme-copper oxidases
(13), the other three subunits
bear no resemblance to subunits of other types of terminal oxidases. However,
subunit CcoO has been shown to have sequence homology with the nitric-oxide
reductase subunit NorC
(14).The crystal structures of a few heme-copper oxidases have been resolved
(15–19),
but only structural homology models are currently available for cytochromes
cbb3
(20–23).
Apart from the signatures common to all heme-copper oxidases, the sequence
alignments have revealed only very few other conserved residues when terminal
oxidases are compared. Even though some amino acids, absent from cytochrome
cbb3, have been shown to be of critical importance to the
function of the classical heme-copper oxidases, the major functions still
remain the same in all of these enzymes.The thermodynamic properties of the cbb3-type oxidases
have been investigated sparsely. Apart from work yielding partial information
about the properties of the hemes
(11,
24,
25), two more complete studies
have been carried out (5,
26). All the hemes in
cytochrome cbb3 were proposed to have high redox
potentials, both in the Pseudomonas stutzeri and Bradyrhizobium
japonicum enzymes (5,
26). This is also the case in
all other studies, except for the enzyme from Rhodothermus marinus,
where two low potential redox centers were reported
(25). However, little is known
about the copper center in the active site (CuB). Early Fourier
transform infrared
(FTIR)2 spectroscopic
measurements identified the presence of a heme/copper binuclear center in
R. sphaeroides cytochrome cbb3
(11), and more recent
resonance Raman and FTIR studies have given additional information about the
structure of the active site
(27–29).In the absence of deconvoluted spectral components and thereby clear
assignments of the redox centers in the cbb3-type
oxidases, and the lack of consensus about their thermodynamic properties, a
complete study was required. In this work we have set out to investigate the
thermodynamic properties of all the redox centers in cytochrome
cbb3 from R. sphaeroides using a combination of
optical and EPR redox titrations with the main focus on the details of the
catalytic site. This effort will form a basis for further mechanistic
studies. |
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