Metal Cluster Active Sites in Proteins

Que

9808350

Proteins with active sites consisting of carboxylate-bridged diiron centers comprise a new subclass of metalloproteins. This growing class includes proteins of various functions such as the dioxygen carrier hemerythrin, a number of hydrocarbon monooxygenases, ribonucleotide reductase, fatty acid desaturases, purple acid phosphatases, and Ser/Thr protein phosphatases. This project concentrates on how the active sites of the different proteins are interrelated and how the metal centers perform their respective functions. Spectroscopic studies on enzymes involved in 2e- oxidation focuses on obtaining the metric parameters for the diiron site in its various oxidation states by EXAFS including catalytically relevant high valent intermediate states. These enzymes include the soluble enzymes, methane monooxygenase, stearoyl acyl carrier protein desaturase, and toluene 4-monooxygenase, as well as the membrane-bound alkane hydroxylase. Resonance Raman studies will be used to characterize intermediates at the diiron(III)-peroxo, diiron(IV)-oxo, and diiron(III)-product states. For ribonucleotide reductase, allosteric effects of the redox potential of the diiron cluster, the source of the 'extra electron' required for tyrosyl radical formation, and the putative diiron(III)-peroxo precursor to the high-valent intermediate X are studied. Physical techniques used in these studies include UV-vis, resonance Raman, EPR, M÷ssbauer, EXAFS, and spectro-electrochemistry, together with the use of mutant proteins to enhance prospects for trapping certain intermediates and for probing long range electron transfer pathways. The purple acid phosphatase from porcine uterus, uteroferrin, will be used as a model for studying the mechanism of dinuclear hydrolases in general and the Ser/Thr protein phosphatases involved in cell signaling in particular. The uncompetitive inhibitor fluoride will serve as a very useful mechanistic probe for the catalytic mechanism, since fluoride very likely substitutes for the nucleophile responsible for phosphate ester hydrolysis. The interaction of fluoride with the enzyme-substrate complex will be investigated by EPR, EXAFS, and resonance Raman techniques to gain insight into the nature of the enzyme-substrate complex prior to hydrolysis. Similar experiments will be carried out on calcineurin and lambda protein phosphatase.

This project is aimed at understanding how metalloenzymes with active sites consisting of two metal centers catalyze metabolically important reactions. For example, methane monooxygenase converts methane to methanol under mild conditions, in contrast to the current energy-intensive industrial process. Ribonucleotide reductase is responsible for making deoxyribonucleotides, the building blocks of DNA, during cell growth. Understanding how to control this enzyme can provide strategies for inhibiting the runaway growth of tumor cells. Cell signaling phosphatases are yet another group of enzymes that use two metal centers; they are important for regulating cellular processes. Using a variety of spectroscopic methods as probes, the active sites of these enzymes in their various forms will be compared to understand their structural relationships, how they catalyze their respective reactions, and the roles of the individual metal centers in carrying out the reactions.