Aerobic organisms gain energy by reducing molecular oxygen to water. Terminal oxidases of the membrane-bound respiratory chains perform the last step of cellular respiration according to the following equation:
O2 + 4e− + 4H+in → 2H2O + ↑nq+
where ↑nq+ stands for endergonic translocation by the oxidases of n elementary charges (mainly protons) across the coupling membrane (positive outside) at the expense of the exergonic redox reaction. In other words, the terminal oxidases work as energy-transducing molecular machines, which pump H+ electrogenically across the membrane at the expense of O2 reduction to water. Given that about 90 percent of biological oxygen consumption in nature occurs via the respiratory oxidases, this is one of life's most fundamental reactions.
The respiratory oxidases fall into two large families: heme-copper oxidases, which are ubiquitous in the living world, from bacteria to humans, and triheme bd-type quinol oxidases, which are widely distributed in the bacterial kingdom but have not been found in eukaryotes. A general aim of our research is to understand the molecular mechanism by which dioxygen reduction to water is coupled to a proton-motive function in different oxidases. The major specific aims of the current project are as follows:
1) We intend to extend our investigations into the molecular mechanism of electrogenic proton translocation by the bacterial cytochrome c oxidase from Rhodobacter sphaeroides, taking advantage of the three-dimensional structure of the enzyme, which was published in 2002 and recently refined to about 2 Å resolution, and the availability of site-specific mutants in virtually all its key amino acid residues.
2) We will explore the possibility that different homologous heme-copper oxidases operate by distinct electrogenic mechanisms. To this end, we plan to devote special attention to the electrogenic mechanism of mitochondrial COX from bovine heart and compare it with those of the aa3-type oxidase from R. sphaeroides and an evolutionarily distant ba3-type COX from Thermus thermophilus, for which the crystal structure is also known.
3) We will compare the oxygen-reduction and electrogenic mechanisms of the proton-pumping oxidases of the heme-copper superfamily with those in the family of the nonpumping triheme bd-type oxidases.
The heme-copper oxidases are so named because their oxygen-reducing center, which is deeply buried inside the membrane, is bimetallic and comprises a high-spin heme and a nearby copper ion (CuB). For each electron transferred to oxygen, one proton is taken up by the heme-copper oxidases to be consumed in water formation (“chemical” proton); in addition, one proton per electron is “pumped”—translocated all the way across the membrane. Accordingly, two electric charges per electron are separated by the "pumping" heme-copper oxidases (n = 2 in the above equation). In recent years, three-dimensional structures have been resolved for the mitochondrial and four bacterial heme-copper oxidases. Each of the structures reveals, besides the redox centers, three intraprotein “pores” opening to the inner side of the membrane that are likely to serve as proton-conducting pathways. These pores are often referred as proton channels D, K, and H after the highly conserved residues Asp, Lys, and His, respectively, which have been shown to be critical for the operation of the three channels.
The specific roles of the three proton-conducting pathways remain under debate. One possibility is that the “chemical” and “pumped” protons may travel by different channels. Alternatively, different channels may be functionally associated with distinct steps in the catalytic cycle. Mutants with replacements of virtually all the conserved amino acid residues within proton channels D and K have been generated for cytochrome oxidase from R. sphaeroides in the laboratory of Robert Gennis at the University of Illinois at Urbana-Champaign, with whom we have had a long collaboration. We will explore the effect of the amino acid replacements in the channels on the kinetics and efficiency (i.e., H+/e− ratio) of intraprotein proton translocation at different steps of the cytochrome c oxidase catalytic cycle. To this end, we will use a unique ultrasensitive electrochemical technique developed earlier in this laboratory by Lel Drachev. The method allows one to monitor electrogenic proton translocation by membrane-bound energy-transducing enzymes with submicrosecond time resolution. We plan to compare the dynamics of vectorial (directed across the membrane) proton transfer in the mitochondrial oxidase, linked to different electron transfer steps of the catalytic cycle, with that of the wild-type and mutant forms of the bacterial oxidases from R. sphaeroidesand Thermus thermophilus. Recent studies in this laboratory and results obtained by other groups have allowed us to outline a provisional route for proton translocation across the membrane through the D-channel coupled to transfer of the fourth electron:
water inside→Asp→...→Glu→…→heme a3-propionate/Arg→H2O-His…→water outside
We hope to trace the entire intraprotein trajectory of the protons translocated by the cytochrome c oxidase from R. sphaeroides at all four redox steps of the four-electron catalytic cycle. Not only will this allow us to understand the specific mechanism of the heme-copper oxidases as the key enzymes of aerobic lifestyle, but it may also help to elucidate the general principles of energy transduction by membrane-bound redox enzymes at the molecular level. Our preliminary observations indicate that the molecular mechanisms of proton translocation in different heme-copper oxidases and, notably, in the bacterial and mitochondrial enzymes, may not be identical. Therefore, we will make a detailed comparison of cytochrome c oxidases from R. sphaeroides and T.thermophilus with the mitochondrial enzyme from bovine heart.
Terminal oxidases of a very different family—cytochrome bd–type oxidases—are widely distributed in prokaryotes and are responsible for the survival of bacteria under various stress conditions—notably, low oxygen concentrations. Expression of cytochrome bd appears to be essential for the virulence of several pathogenic bacteria, including Mycobacterium tuberculosis. Cytochrome bd does not contain copper but carries three hemes: one low-spin (b558) and two high-spin (b595 and d). The enzyme catalyzes the same redox reaction as the heme-copper oxidases. However, it does so two to three times faster but conserves energy with half the efficiency (n = 1 in the first equation); it does not pump protons and takes up only the “chemical” proton required for water formation. Hence, cytochrome bd–type oxidases perform transmembrane separation of only one electric elementary charge pair per electron. So far, no three-dimensional structure has become available for any bd-type oxidase. Using several spectroscopic techniques as well as the time-resolved electrometric method, we will investigate the rapid kinetics of intraprotein electron and proton transfer for wild-type and mutant forms of cytochrome bd from Escherichia coli and Azotobacter vinelandii. We will test the hypothesis that bd oxidases contain a bimetallic, oxygen-reducing center formed by two high-spin hemes (d and b595) instead of by heme and copper. Comparing the redox and electrogenic mechanisms of the pumping heme-copper oxidases with those in nonpumping bd-type oxidases lacking copper in the oxygen-reducing site may be essential for understanding the specific role played by the CuB ion in proton pumping by the heme-copper oxidases. Finally, given that bd-type oxidases are confined to bacterial respiratory chains, specific inhibitors of cytochromes bd may provide a promising basis for the development of antibacterial drugs.
Last updated September 2008
