Biochem - Hille, Russ

My research program focuses the reaction mechanisms of oxidoreductase enzymes - particularly those possessing molybdenum or flavin in their active sites - and biological electron transfer. The molybdenum-containing enzymes catalyze the incorporation of oxygen into a variety of organic and inorganic compounds, and constitute an important enzyme class within the oxidoreductases. These enzymes are only poorly understood in comparison to other biological systems that contain heme, flavin, non-heme iron or copper. Working with representative members of each of the three major families of molybdenum enzymes, we have successfully identified the fundamental aspects of the catalytic sequences of these enzymes. Particularly in the case of the molybdenum hydroxylase family (as represented by xanthine oxidase), work in our laboratory has elucidated the chemical course of the reaction as well as the role of specific active site residues in accelerating reaction rate. This work has demonstrated that molybdenum-catalyzed hydroxylation of carbon centers occurs without the generation of a highly reactive oxygenated intermediate (e.g., the perferryl oxide and 4a-peroxide "oxygen guns" of heme and flavin containing enzymes, respectively). Our present work in this area involves kinetic, spectroscopic and mutagenic studies to gain further insight into the basic elements of catalysis at play in these enzymes, as well as modeling of the reaction intermediates and transition states using computational approaches.

Several of the enzymes we are investigating possess multiple redox-active centers within a single polypeptide, and are useful systems in which to examine the factors governing rates of biological electron transfer without the complication of protein-protein interactions. We utilize pH-jump stopped-flow, flash photolysis and pulse radiolysis methodologies to examine the rates of electron transfer within several such enzymes, including xanthine oxidase (possessing a molybdenum center, two [2Fe-2S] clusters and FAD), trimethylamine dehydrogenase (with FMN and a [4Fe-4S] cluster) and succinate:quinone oxidoreductase (with FAD, three different iron-sulfur centers, a b-type cytochrome and a tightly bound equivalent of ubiquinone). We find, for example, that protonation/deprotonation events are frequently tightly coupled to electron transfer, although electron transfer is generally extremely rapid and not rate-limiting for catalytic turnover. Our present studies are focused on elucidating specific pathways of electron transfer within these and other systems, particularly with regard to understanding the role of branches in these pathways and the function of sites that possess "outlier" reduction potentials – either very high or very low – in these simple electron transport chains.

Hemann, C.F., Ilich, I., Stockert, A.L., Choi, E.-Y., & Hille, R. (2005) Resonance Raman studies of xanthine oxidase: the reduced enzymeoproduct complex with violapterin. J. Phys. Chem. B. 109, 3023-3031.

Wei, C.C., Wang, Z.Q., Durra, D., Hemann, C., Hille, R., Garcin, E.D., Getzoff, E.D., and Stuehr, D.J. (2005) The three nitric-oxide synthases differ in their kinetics of tetrahydrobiopterin radical formation, heme-dioxy reduction and arginine hydroxylation. J. Biol. Chem. 280, 8929-8935.

Cobb, N., Conrads, T., & Hille, R. (2005) The reaction of reduced dimethylsulfoxide reductase with dimethylsulfoxide. J. Biol. Chem. 280, 11007-11017.

Doonan, C.J., Stockert, A.L., Hille, R., & George, G.N. (2005) Nature of the catalytically labile oxygen in the active site of xanthine oxidase. J. Am. Chem. Soc. 127, 4518-4522.

Shi, W., Mersfelder, J., & Hille, R. (2005) The interaction of trimethylamine dehydrogenase with electron-transferring flavoprotein. J. Biol. Chem. 280, 20239-20246.

Anderson, R.F., Hille, R., Shinde, S. and Cecchini, G. (2005) Electron transfer with succinate:ubiquinone oxidoreductase (SQR) of Escherichia coli. J. Biol. Chem. 280, 33331-33337.

Astashkin, A.V., Hood, B.L., Feng, C., Hille, R., Mendel, R.R., Raitsimring, A.M., & Enemark, J.H. (2005) Studies of the Mo(V) forms of sulfite oxidase from Arabidopsis thaliana by pulsed EPR. Biochemistry 44, 13274-13281.

Stuehr, D.J., Wei, C.C., Wang, Z.Q., and Hille, R. (2005) Exploring the redox reactions between heme and tetrahydrobiopterin in the nitric oxide synthases. Dalton Trans. 21, 3427-3435.

Hemann, C.F., Hood, B.L., Fulton, M., Hänsch, R., Mendel, R.R., Kirk, M.L., & Hille, R. (2005) Spectroscopic and kinetic properties of sulfite oxidase from Arabidopsis thaliana: nature of the redox-active orbital and electronic structure contributions to catalysis J. Am. Chem. Soc. 127, 16567-16577.

Pei, P., Horan, M.P., Hille, R., Hemann, C.F., Schwendeman, S.P., & Mallery, S.R. (2006) Reduced nonprotein thiols inhibit activation and function of MMP-9. Implications for chemoprevention. Free Rad. Biol. Med. 41, 1315-1324.

Kundu, T.K., Hille, R., Velayutham, M., & Zweier, J.L. (2007) Characterization of superoxide production from aldehyde oxidase: an important source of oxidants in biological tissues. Arch. Biochem. Biophys. 460, 113-121.

Pauff, J.L., Hemann, C.F., Leimkühler, S., and Hille, R. (2007) The role of arginine 310 in catalysis and substrate specificity in xanthine dehydrogenase from Rhodobacter capsulatus. J. Biol. Chem 282, 12785-12790.

Astashkin, A., Johnson-Winters, K., Klein, E.L., Byrne, R.S., Hille, R., Raitsimring, A.M., & Enemark, J.H. (2007) Direct demonstration of the presence of coordinated sulfate in the reaction of Arabidopsis thaliana sulfite oxidase using 33S-labeling and ESEEM spectroscopy. J. Am. Chem. Soc. In press.

Erzurum, S.C., Ghosh, S., Janocha, A.J., Xu, W., Bbauer, S., Bryan, N.S., Tejero, J., Hemann, C., Hille, R., Stuehr, D.J., Geelisch, M., and Beall, C.M. (2007) Higher blood flow and circulating nitric oxide products offset high-altitude hypoxia among Tibetans. Proc. Natl. Acad. Sci. USA 104, 17593-17598.

Cobb, N., Hemann, C.F., Polsinelli, G., Ridge, J.P., McEwan, A.G., & Hille, R. (2007) Spectroscopic and kinetic studies of Y114F and W116F mutants of DMSO reductase from Rhodobacter capsulatus. J. Biol. Chem., 282, 35519-35529.

Pauff, J.M., Zhang, J., Bell, C.E., and Hille, R. (2007) Substrate orientation in xanthine oxidase. The crystal structure of an intermediate in the reaction with 2-hydroxy-6-methylpurine. J. Biol. Chem. 282, 35519-35529.