Molecular architecture of UQH2:cyt c2 oxidoreductase

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DESCRIPTION (provided by applicant): We propose to further investigate the mechanism of the bc1 complex (UQH2:cyt c2 oxidoreductase) in order to understand its role in cellular aging, and its function as the target for drugs and pest-control reagents. These latter depend on differential sensitivities to quinone-mimics that act as anti-malarial drugs, fungicides, pesti- cides, herbicides, etc., in different species. These enzymes are at the core of all major respiratory and photosynthetic pathways, and are directly responsible for about 30% of the energy conversion of the biosphere. This central importance in biology provides an intrinsic interest, relating directly to our understanding of cellular physiology, energy conversion mechanisms, and maintenance. The photosynthetic bacteria provide a model system for studying the medically important mitochondrial complex. The catalytic core of the bc1 complex is highly conserved across the mitochondrial-bacterial divide, and the reaction mechanism is essentially the same. In the bacterial system, the interplay between function and structure can be more easily studied because the system can be activated by illumination, initiating turnover in the 10 <s time scale. In addition, the bacte- rial system is readily amenable to molecular engineering through specific mutagenesis. The research supported by this grant has contributed substantially to our understanding of how these complexes function. We take advantage of 35 years of experience in assaying function to explore the mechanism through a multi-pronged approach exploiting the synergy between molecular engineering, biophysical assay, structural studies by X-ray crystallography, detailed analysis of local structure through spectroscopy, and modeling through computer simulation. The molecular architecture of the complex that is emerging from these studies provides one of the most detailed descriptions of a molecular machine of this complexity currently available. The availability of crystallographic structures has stimulated much interest, and has provided strong support for the modified Q- cycle we proposed, which is generally accepted as the underlying mechanism. However, the structures have also provoked some interesting questions, mainly relating to unexpected dynamic features, including a large scale domain movement, and a more subtle local molecular ballet that allows rapid turnover without damaging bypass reactions. In this proposal, we address some of the more controversial issues, including features of the mechanism that minimize production of precursors of the damaging reactive oxygen species that lead to cellular suffocation. The proposal is for continuation of work on one of the key enzymes of metabolism, the bc1 complex (ubihydroquinone -cytochrome c oxidoreductase). Mitochondria power the cell through oxidation of metabolites, using the respiratory chain to pass electrons to O2. The bc1 complex is the central enzyme of the chain. A design defect from its evolutionary past has left this complex with an ability to generate damaging oxygen radicals that harm the cell. We study the same enzyme in Rhodobacter sphaeroides, a photosynthetic bacterium close to the bacterial ancestor of the mitochondria. Because the enzyme can be activated through the photosynthetic machinery, it is much easier to study rapid, single-turnover kinetics, and hence to probe the mechanism. The bacterial system has become a standard experimental model for this important enzyme. By understanding the mechanism, we hope to understand how the damaging radicals are generated, and how evolution has fined-tuned the mechanism so as to minimize this reaction. The complex is also a target for anti- malarial drugs, and for fungicides and pesticides, important both in medicine and agriculture.