Function of the Cofactors of Complex II from Escherichia coli

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Gary Cecchini MCB-9728778 1. Technical Abstract This study is aimed to investigate the function of the cofactors of fumarate reductase (menaquinol-fumarate oxidoreductase) (QFR) and succinate dehydrogenase (succinate-ubiquinone oxidoreductase) (SQR) from Escherichia coli. These two membrane-bound enzyme complexes, found in most prokaryotes and eukaryotes, are structurally and genetically related and are important components of anaerobic and aerobic respiratory chains, respectively. The cofactors of the enzyme include a covalently bound FAD cofactor, bound to the largest membrane extrinsic subunit of the complex which contains the substrate binding site. The iron-sulfur containing subunit of the complex contains three distinct Fe-S clusters. The Fe-S clusters are: Center 1, a 2Fe-2S 2+,1+ cluster; Center 2, a 4Fe-4S 2+,1+ cluster; and Center 3, a 3Fe-4S 1+,0 cluster. The membrane intrinsic domain of the complexes contain two hydrophobic subunits which provide site(s) for the interaction of the enzyme with quinone cofactors. In addition, the SQR complex, (but not QFR) contains a heme prosthetic group. The enzyme complexes can catalyze the same reactions with both SQR and QFR proficient at succinate oxidation, however, only QFR is highly active as a fumarate reductase. The nature of electron transfer through the highly similar enzyme complexes and their cofactors must therefore be responsible for the inability of SQR to efficiently reduce fumarate in catalytic assays. The reasons for this difference will be a major focus of this proposal. Experiments are designed to use biochemical, biophysical, and molecular biological techniques to investigate the protein environment responsible for the functioning of the FAD, Fe-S, and heme cofactors in the enzymes. The enzyme complexes are easily manipulated by site-directed mutagenesis and both wild type and mutant forms of the enzyme can be isolated in very high yield. The large amounts of highly purified enzyme complex which is produced aids in EPR, magnetic circular dichroism (MCD), and resonance Raman spectroscopic methods used to analyze the environment of the Fe-S clusters and the redox potential of these important cofactors. Site-directed mutants of both enzyme complexes will be constructed and analyzed for their effect on the Fe-S clusters. This will be done to determine if the redox potential of the Fe-S clusters can be manipulated in a predictable manner and if the differences in redox potential for the Fe-S clusters of SQR and QFR are the major reason for the differences in the catalytic activity between the two enzyme complexes. Investigations will also be begun to determine if time resolved kinetic measurements of electron transfer through the various cofactors of QFR and SQR can be measured. This will be accomplished by following the photooxidation of pyranine dyes placed specifically at the active site of SQR and QFR and monitoring electron transfer through the FAD, heme, and quinone cofactors of the enzymes. These studies will aid in the understanding of electron transfer reactions through proteins. Important information will also be provided on the differences in electron transfer through the highly similar QFR and SQR enzyme complexes. Gary Cecchini, Ph.D. MCB-9728778 2. Non-technical abstract This study investigates the function of the cofactors of two enzyme complexes important for the function of respiratory function in cells in both an aerobic and anaerobic environment. The enzyme complexes studied are fumarate reductase (menaquinol-fumarate oxidoreductase, QFR) and succinate dehydrogenase (succinate-ubiquinone oxidoreductase, SQR) usually from the bacterium Escherichia coli. These two membrane-bound enzyme complexes, found in most prokaryotes and eukaryotes, are structurally and genetically related. Both enzyme complexes contain a covalently bound FAD cofactor, bound to the largest membrane extrinsic subunit of the complex, which also contains t he dicarboxylate binding site. Each enzyme also contains three distinct iron-sulfur clusters which participate in the electron transfer function of the enzymes. The iron-sulfur clusters have distinct redox potentials spanning a range of several hundred mV. In addition, to the iron-sulfur cluster the SQR complex, but not the QFR complex, contains a heme cofactor as an additional prosthetic group. The major questions being investigated are what factors in the protein environment control the redox potential of the various prosthetic groups in these two enzymes and thus the path of electron flow through the complexes. Under defined conditions succinate dehydrogenase acts as a "tunnel diode" in directing the path of electrons through the various prosthetic groups which act as the electron wires in the protein complex. Using the techniques of site-directed mutagenesis and electron paramagnetic resonance spectroscopy (EPR) the protein environment will be modified to understand how one can alter the rate and directionality of electron flow in these enzymes by modification of the redox potential of the cofactors. These studies will allow a more complete understanding of ways nature designs the micro protein machinery involved in electron transfer reactions. In addition, the studies will show how two very similar enzyme complexes can be poised to catalyze different chemical reactions.