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. This multi-subunit electron transfer complex contains cytochrome b, which contains two heme groups, cytochrome c1, and an iron-sulfur protein containing a 2Fe: 2 S 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. The crystal structure of the chicken enzyme can be viewed at Ed Berry's Web Site. The bc1 complex from yeast was crystallized by Carola Hunte, who is at the Max Planck Institute for Biophysics.
We are investigating aspects of the mechanism of the enzyme and the process by which the enzyme is assembled into the inner mitochondrial membrane. We are 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. There are numerous opportunities available for post-doctoral research. These positions provide training in protein biochemistry, enzyme kinetics, yeast molecular biology, and molecular modeling.
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-c ubiquinol forms an electron donor complex by binding to the Rieske protein and cytochrome b and ubiquinol oxidation delivers two electrons divergently to the Rieske iron-sulfur cluster and the bL heme. The resulting ubiquinone leaves center P. In reaction 2 the reduced Rieske cluster moves to within electron transfer distance of 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 while a second proton is released through a pathway on cytochrome b. In reaction 3 an electron is transferred from the bL to bH heme, which in turn reduces ubiquinone to ubisemiquinone (reaction 4). Following oxidation of a second ubiquinol at center P by repeat of reactions 1-3, the bH heme reduces ubisemiquinone to ubiquinol (reaction 5), accompanied by uptake of two protons at center N.
Although the Q cycle mechanism is well established, several details of the mechanism are not fully understood. We are investigating the role of the Rieske iron-sulfur protein in oxidation of ubiquinol and the mechanism of ubisemiquinone participation in electron transfer at the ubiquinone reduction site in the bc1 complex. 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).
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). The concerted, alternating sites mechanism has been summarized in a recent refinement of the Q cycle (9). We are now attempting to understand how the two bc1 monomers cooperate in the dimer.
We are also investigating the mechanism by which hydroxyquinones bind to the ubiquinol oxidation site at center P in the yeast bc1 complex. Hydroxyquinones, 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 hydroxyquinones 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 (15) and how mutations in cytochrome b have caused atovaquone resistance in Pneumocystis jirovecii (16). A post-doctoral position is available to continue these studies in Plasmodium falciparum, the parasite that causes malaria.
Assembly of the cytochrome bc1 complex
The cytochrome bc1 complex in yeast consists of 10 subunits. All of the subunits except cytochrome b are encoded in the nucleus, synthesized in the cytoplasm, transported into mitochondria, and assembled into the mitochondrial membrane. Cytochrome b is encoded in the mitochondrial genome and forms the hydrophobic core of the enzyme, around which the nuclear encoded subunits are assembled. The crystal structure of the cytochrome bc1 complex shows the enzyme to be dimeric and is a useful guide to subunit interactions that might be anticipated during intermediate steps in the assembly of this enzyme complex.
The questions that we are attempting to answer have broader implications for assembly of all oligomeric membrane proteins. What is the temporal and mechanistic relationship between import of subunits into mitochondria, prosthetic group insertion, and subunit assembly into the inner membrane enzyme complex? Are subunits assembled by a linear pathway in a sequential manner, or by a convergent pathway involving intermediate sub-complexes? Does assembly of the bc1 complex begin with co-assembly with the cytochrome c oxidase and/or ATPase complexes in which the mitochondrial encoded proteins of these complexes form a common nucleation core? Are there chaperones for assembly of the bc1 complex and, if so, are they specific for assembly of this membrane protein complex or generic? At what point during assembly does the enzyme become dimeric, and what, if any, chaperones are required for dimerization?
Results from our initial experiments in this area, which have focused on import of the Rieske iron-sulfur protein and subunit 6 , are described in the papers listed below. These two subunits of the bc1 complex are similarly located on the intermembrane surface of the enzyme, but are imported by different mechanisms. The iron-sulfur protein contains a presequence that is cleaved during import. The presence of this presequence is essential for import, but import occurs under conditions where cleavage of the presequence is blocked, showing that the two steps are not obligatorily linked (10 ). One of the interesting features of the iron-sulfur protein presequence is that during import it is cleaved in two steps in S. cerevisiae but cleaved in only one step in numerous other species, including mammals. We showed that this two step processing is not essential for import in yeast (11) and that exchange of a proline for a serine residue in the presequence is responsible for the difference between one step and two step processing (12). In our studies on import of the iron-sulfur protein we showed that the intermediate length iron-sulfur protein that is formed by the first proteolytic processing step is present in the bc1 complex and active (13). This result demonstrates that conversion of intermediate to mature length occurs after the iron-sulfur protein is assembled into the bc1 complex and that the 2 Fe:2 S cluster is inserted before the intermediate is processed to mature size.
Subunit 6 of the bc1 complex also has a presequence that is cleaved when the protein is imported into mitochondria. However, the presequence is not required for its import in vivo or in vitro and is also not required for assembly of this subunit into the bc1 complex (14). Information for targeting and import of this subunit appears to be present in the C-terminus of the protein. The mechanism by which subunit 6 is imported into mitochondria and assembled into the bc1 complex is still under investigation.
In our current studies on assembly of the bc1 complex we are focusing on isolation of intermediate assembly complexes. Our objective is to deduce the complete assembly pathway and to identify factors that assist in assembly of this oligomeric enzyme complex into the inner mitochondrial membrane.
8. Emma Berta Gutierrez-Cirlos and Bernard L. Trumpower, (2002) "Inhibitory Analogs of Ubiquinol Act Anti-cooperatively on the Yeast Cytochrome bc1 Complex. Evidence for an Alternating, Half-of-the-Sites Mechanism of Ubiquinol Oxidation," J. Biol. Chem. 277, 1198-1202
10. Nett, J. H. and Trumpower, B. L. (1996) "Dissociation of Import of the Rieske Iron-Sulfur Protein into Saccharomyces cerevisiae Mitochondria from Proteolytic Processing of the Presequence," J. Biol. Chem. 271, 26713-26716
11. Nett, J. H., Denke, E. and Trumpower, B. L. (1997) "Two Step Processing is Not Essential for the Import and Assembly of Functionally Active Iron-Sulfur Protein into the Cytochrome bc1 Complex in Saccharomyces cerevisiae," J. Biol. Chem. 272, 2212-2217
12. Nett, J. H. and Trumpower, B. L. (1998) "Processing of the Presequence of the Schizosaccharomyces pombe Rieske Iron-Sulfur Protein Occurs in a Single Step and Can be Converted to Two Step Processing by Mutation of a Single Proline to Serine in the Presequence," J. Biol. Chem. 273, 8652-8658
13. Nett, J. H. and Trumpower, B. L. (1999) "Intermediate Length Rieske Iron-Sulfur Protein is Present and Functionally Active in the Cytochrome bc1 Complex of Saccharomyces cerevisiae," J. Biol. Chem. 274, 9253-9257
14. DeLabre, M. L., Nett, J. H. and Trumpower, B. L. (1999) "The Cleaved Presequence is not Required for Import of Subunit 6 of the Cytochrome bc1 Complex into Yeast Mitochondria or Assembly into the Complex," FEBS Letts. 449, 201-205