Overview
The members of the Trumpower laboratory are conducting research on the mitochondrial cytochrome bc1 complex. The cytochrome bc1 complex is a multi-subunit electron transfer complex found in the inner membrane of mitochondria, the plasma membrane of many bacteria, and the thylakoid membrane of chloroplasts. The bc1 complex is especially important in cell respiration since it is a confluence point for electrons entering the respiratory chain from the various dehydrogenases. The bc1 complex is also one source of superoxide anion, the reactive oxygen species that is produced as an aberrant by-product and that has been implicated in tissue damage in various diseases and the aging process. This multi-subunit electron transfer complex contains cytochrome b, which contains two heme groups, cytochrome c1, and an iron-sulfur protein containing a 2Fe:2S cluster. In mitochondria and chloroplasts the bc1 complex is essential for respiration and photosynthesis, respectively. In bacteria the bc1 complex is involved in respiration, photosynthesis, nitrogen fixation, and denitrification, depending on the type of bacteria.The bc1 complex has been crystallized from bovine, chicken, and yeast mitochondria and from Rhodobacter. We are investigating the mechanism of the enzyme and the structural basis of the mechanism, using the yeast Saccharomyces cerevisiae as the organism for our experiments, since this allows a combination of experimental approaches from biochemistry, genetics, and molecular biology.
Mechanism of the cytochrome bc1 complex
The mitochondrial cytochrome bc1 complex transfers two electrons from ubiquinol to two molecules cytochrome c. As two electrons are transferred, four protons are deposited at the outer surface of the inner membrane while two protons are taken up from the inner, matrix surface of the membrane. In this manner the energy released from the electron transfer reaction is captured in the form of a transmembrane electrochemical proton gradient. This gradient is then used for energy requiring processes such as ATP synthesis, ion transport, and flagellar movement. A similar electron transfer and proton translocation is catalyzed by the analogous enzyme located in the plasma membrane of bacteria and in the thylakoid membrane of chloroplasts.
The mechanism by which the bc1 complex transfers electrons from ubiquinol to cytochrome c and links electron transfer to proton translocation is generally understood. This mechanism, shown in Figure 1, is known as the protonmotive Q cycle (1,2).
Figure 1 The protonmotive Q cycle. Circled numbers designate electron transfer reactions. Dashed arrows designate movement of ubiquinol (QH2) or ubiquinone (Q) between center P and center N and movement of the iron-sulfur protein between cytochrome b and cytochrome c1. Black bars indicate sites of inhibition by antimycin, funiculosin, ilicicolin, myxothiazol, stigmatellin and UHDBT. In reaction 1a ubiquinol forms an electron donor complex by binding to the Rieske protein (QH2ISP) and ubiquinol oxidation delivers two electrons in a bifurcated reaction (1 b,c) to the Rieske iron-sulfur cluster and the bL heme. Whether this bifurcated reaction is concerted or sequential is one of the questions addressed in our research. In reaction 2 the reduced Rieske protein moves proximal to cytochrome c1, resulting in electron transfer from the iron-sulfur cluster to the c1 heme and release of one proton from the Rieske protein at center P. A second proton is released coincident with quinol oxidation through a pathway on cytochrome b (reaction 1b,c). Identifying this pathway is another aim of our research. In reaction 3 an electron is transferred from the bL to bH heme, which reduces ubiquinone to ubisemiquinone at center N (reaction 4). Following oxidation of a second ubiquinol at center P (reactions 1-3), the bH heme reduces ubisemiquinone to ubiquinol (reaction 5), accompanied by uptake of two protons at center N.
Current Research
Although the Q cycle mechanism is well established, several details of the mechanism are not fully understood. In our studies on the Rieske iron-sulfur protein we showed that the mid-point potential of the Rieske protein is quantitatively the most important parameter in determining the rate of electron transfer though the bc1 complex (3, 4). We also showed that changing the length of the flexible linker region that connects the iron-sulfur cluster domain to the transmembrane anchor domain results in loss of ubiquinol oxidation activity, which we attribute to changes in the structure of the ubiquinol oxidase site that is formed between the iron-sulfur protein and cytochrome b (5). By examining the pre-steady state reduction of cytochrome b in the cytochrome bc1 complex from a yeast mutant that lacks endogenous ubiquinone and in bc1 complex from wild-type yeast in the presence of antimycin, we showed that ubiquinone residing at center N in the oxidized bc1 complex is responsible for the triphasic reduction of cytochrome b (6). We have proposed that oxidation of ubiquinol at center P occurs by a concerted mechanism, based on our findings that there is reciprocal control on the presteady state reduction of the high potential and low potential redox centers of the bc1 complex (7). One of the aims of our current research is to further test whether ubiquinol oxidation is a concerted reaction, or a sequential reaction involving a semiquinone intermediate.
We have recently shown that binding of inhibitory ubiquinol analogs at the quinol oxidase site in the bc1 dimer is anti-cooperative, in that binding of inhibitor at center P in one monomer markedly reduces the affinity for binding of a second molecule of inhibitor or substrate at center P in the other monomer. Since these inhibitors are generally considered to mimic a transition state in ubiquinol oxidation, we have proposed that the structurally dimeric bc1 complex is functionally dimeric, and that ubiquinol oxidation occurs by an alternating, half-of-the-sites mechanism (8, 16). The concerted, alternating sites mechanism has been summarized in a recent refinement of the Q cycle (9). The crystal structures have shown that both mitochondrial and bacterial bc1 complexes are structural dimers. We recently discovered that the dimeric structure allows for an extensive set of regulatory interactions that involve communication between center N and center P across the inner mitochondrial membrane. We have suggested that the dimeric structure was selected very early and retained during evolution to minimize the formation of reactive oxygen species and maintain maximum turnover rates as the ubiquinone/ubiquinol redox potential changes (29). One unusual feature of the dimeric structure is that the Rieske protein is anchored by a trans-membrane helix in one monomer, while the iron-sulfur cluster domain of the protein functions in the second monomer. The structure of the Rieske protein spanning the two halves of the dimer suggests that it may have a role in the recently discovered regulatory interactions between the two monomers (29, 41).
Following the proposal of a concerted, alternating sites mechanism of ubiquinol oxidation (8, 16), we showed that inhibitory analogues of ubiquinol bind anti-cooperatively at center P (8) and that ubiquinol oxidation is also anti-cooperative (25). This supported the premise that center P exhibits half-of-the-sites reactivity. More recently, we discovered that center N also manifests half-of-the-sites reactivity, that this reactivity is regulated by ligands that bind to center P (12), and that there is rapid electron transfer between monomers in the bc1 dimer (27, 40). These findings indicate that the regulatory interactions in the bc1 dimer are much more extensive than we originally envisioned when we discovered the half-of-the-sites reactivity at center P. One of our current research goals is to elucidate the structural basis of the regulatory interactions between the two monomers that is responsible for the anti-cooperativity at center N and center P and the structural basis of the regulatory interaction between the ubiquinol oxidation and ubiquinone reduction centers in the enzyme. This is an especially interesting question, since center N and center P are ~35 Å apart, on opposite sides of the mitochondrial membrane.
We are also investigating the mechanism by which hydroxynaphthoquinones bind to the ubiquinol oxidation site at center P in the yeast bc1 complex. These hydroxyquinones are competetive inhibitors of the enzyme, and thus their binding must mimic the binding of ubiquinol to the quinol oxidation site at center P in the bc1 complex. Understanding how these inhibitors are bound will help us better understand the non-covalent interactions responsible for binding ubiquinol. In addition, hydroxynaphthoquinones, such as atovaquone, are used to treat Plasmodium falciparum, Pneumocystis jirovecii, and numerous other parasitic and fungal infections. The usefulness of these bc1 inhibitors as therapeutic agents is being compromised by the emergence of spontaneously arising mutations conferring resistance. Many of the resistance alleles have been mapped to and sequenced on the mitochondrial cytochrome b gene. We are recapitulating these mutant alleles in Saccharomyces cerevisiae, so that we can investigate the interaction of the hydroxynaphthoquinones with the mutant enzyme in a non-pathogenic model fungus, for which a crystal structure is available. We have been able to describe how atovaquone binds to the bc1 complex (20) and how mutations in cytochrome b have caused atovaquone resistance in Plasmodium falciparum (26) and Pneumocystis jirovecii (23).