The objective of this application is to elucidate the structure, function, and mechanism of the ubiquinone (Q)-mediated electron transfer complexes of the mitochondrial respiratory chain. During past support periods the applicant has established protocols for site-directed mutagenesis coupled with in vivo and in vitro reconstitution to study some of the structural and functional relationship in succinate-Q reductase (SQR) and ubiquinol-cytochrome c reductase (QCR). He has crystallized QCR, solved its structure to 2.9 A resolution and indicated that QCR is a functional dimer and that the catalytic cycle of complex involves movement of the head domain of iron-sulfur protein (ISP). Functional evidence for such movement was obtained from characterization of Rhodobacter sphaeroides bc1 complexes with altered ISP necks. In addition to electron and proton transfer, this group has discovered and studied two more functions of QCR, superoxide radical generation and mitochondrial processing peptidase activity (MPP). In the next grant period Dr.Yu will focus on the elucidation of the mechanism of electron transfer and proton translocation in QCR and of electron transfer in SQR. The approaches to be used include kinetic rate determinations, various spectroscopic measurements, organic synthesis, resolution and reconstitution, molecular genetics, and protein crystallography.
The specific aims are: (A) to obtain structural data for mechanistic studies, including the Q-binding at the Qo site pocket, the proton transfer path, structure water, hydrogen bonding, and the interaction domain between cytochrome c1 and c. This will be achieved by obtaining high resolution QCR structure and by X-ray analysis of QCR crystals in different redox states, in the presence of non-oxidizable Q derivatives, or with cytochrome c; (B) to elucidate the reaction mechanisms for electron and proton transfer in QCR. This will be achieved by investigating the movement of the head domain of ISP via functional analysis of mutants having an intersubunit cystine bridge, salt bridge, or hydrogen bond at the interface between cytochrome b and ISP, and via the conformational changes of cytochrome b or c1, at the interface with ISP, during the oxidation-reduction cycle or binding with different Qo site inhibitors; by determining the acid induced intra-molecular electron transfer rates between 2Fe2S and heme c1 using proton caged compound activated by pulse laser; and probing the nature of the intra-molecular path of QH2 in QCR by site directed mutagenesis; (C) to synthesize Q-derivatives and inhibitors for protein:Q interaction studies; (D) to elucidate electron transfer mechanisms in SQR by establishing the identity of QPs2; reconstitution of SQR from recombinant QPs subunit and isolated SDH; identification of SDH docking site(s) in QPs subunits; and crystallization and 3-D structural analysis of mitochondrial SQR. Successful elucidation of the reaction mechanisms for electron and/or proton transfer of SQR and QCR will provide information crucial to understanding the mitochondrial bioenergetic process and thus help in drug design for mitochondrial related diseases. Also, since the quinone reactive sites are responsible for superoxide generation and Q can scavenge singlet oxygen, detailed knowledge of the structure-function relationships and reaction mechanisms of Q-mediated electron transfer should provide valuable information for the study of oxidative stress. This information will be useful in pharmaceutical investigations of cytotoxicity and the aging process.
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