Metalloprotein Structure, Function & Mechanism

dmdooleymontana.edu

My group is conducting research on the structures, reactivities, regulation, biosynthesis, and functions of several metalloenzymes as well as other areas of bioinorganic chemistry. Copper-containing amine oxidases are widely distributed in nature and are involved in the metabolism of biogenic primary amines, which have a variety of functions in the cardiovascular, gastrointestinal, and nervous systems. Amine oxidases are also responsible for the cross-linking of connective tissue structural proteins (elastin and collagen). Recently, amine oxidases have emerged as the first examples of what may prove to be a wholly new class of enzyme, that is, where a post-translationally modified amino acid side chain is present in the active site and has a redox role in catalysis. The principal goals of our research are to elucidate the molecular structures, the catalytic mechanisms, and the mechanism of post-translation modification of copper-containing amine oxidases. Coordinated structural, spectroscopic, and mechanistic experiments are carried out concurrently. We utilize a wide variety of spectroscopic techniques including magnetic circular dichroism, x-ray absorption (EXAFS), resonance Raman, and cw and pulsed epr. The structure of amine oxidases at atomic resolution are being determined in collaboration with x-ray crystallography groups. The formation and subsequent reactions of intermediates are investigated by both temperature-jump and stopped-flow absorption/circular dichroism. Additional mechanistic information is derived from spectroscopic and kinetics experiments with substrates and reactions with substrate analogues. Comparative studies on amine oxidases, methylamine dehydrogenases, and galactose oxidase provide additional insights into structure-function relationships among enzymes containing post-translationally modified, redox-active amino acids in their active sites. We have recently demonstrated that the two processing events which occur in the maturation of galactose oxidase, the cleavage of a 17 amino acid N-terminal pro-sequence and the formation of the Tyr-Cys cofactor, are self-processing reactions requiring only copper ions and dioxygen.

Keywords:
Bioinorganic, Biochemistry

Copper-containing enzymes are also involved in denitrification (the reduction of nitrate to nitrogen), which is a key component of the global nitrogen cycle. Extensive structural, spectroscopic, and chemical studies on nitrate reductase and nitrous oxide reductase are being pursued. These two classes of multicopper redox enzymes provide new opportunities to investigate structure/function relationships in copper-containing enzymes that are involved in denitrification. The principal goal of our research is to achieve a fundamental understanding of the structural and catalytic roles played by the metal ions and protein moieties in nitrate and nitrous oxide reductases. Questions concerning the possible regulation of these enzymes by intermediates in the denitrification pathway are also addressed. The intrinsic spectroscopic properties of Cu(II) are exploited as a built-in probe of the active site by variable temperature x-ray absorption (EXAFS), absorption, circular dichroism, magnetic circular dichroism, resonance Raman, and EPR spectroscopy. Some of the copper sites in the nitrite and nitrous oxide reductases are closely related to copper sites in other multicopper metalloenzymes. One of our principal findings is that nitrous oxide reductase contains CuA-type sites, which were first identified in cytochrome c oxidase. We have also identified a novel copper-sulfide cluster in the catalytic site of this enzyme. This is the first demonstration of a Cu4S cluster in chemistry and biochemistry. Integrating results from the proposed experiments and mechanistic studies will permit new structure/reactivity/function correlations among multi-copper oxidases and reductases to be formulated.

Selected Publications

Chan, JM, Bollinger, JA, Grewell, CL, and Dooley, DM :
Reductively activated nitrous oxide reductase reacts directly with substrate
JACS, 126, 3030-3031 (2004)

Alvarez, ML, Ai, J, Zumft, W, Sanders-Loehr, J, and Dooley, DM. :
Characterization of the copper-sulfur chromophores in nitrous oxide reductase by resonance raman spectroscopy: Evidence for sulfur coordination in the catalytic cluster.
JACS, 123, 576-587, (2001)

Keywords:
Structure, Spectroscopy, Protein Chemistry, Mechanism, Inorganic, Bioinorganic

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