Many essential biological processes involve electron-transfer either between protein partners or between protein partners within multicomponent enzyme complexes where electron transfer at the catalytic center may be coupled to chemical reactions. Examples of both types of electron transfer include the photosynthetic reduction of NADP+ to NADPH; the synthesis of deoxyribonucleotides; the oxidation of methane; and the (3-oxidation of fatty acids. In each of the above cases, recent thermodynamic and in some cases kinetic measurements of the important protein components have suggested that electron transfer, and thus the overall reaction mechanisms, are regulated by the binding of protein partners, enzyme components, or substrates/products. We have found that the redox properties of the cofactors of proteins are excellent reporter groups for changes occurring in a protein upon binding of regulatory subunits or substrates; sometimes changes are observed with this method which cannot be observed spectrally. From our redox studies, we have discovered two very different and interesting cases of regulation of electron transfer reactions coupled to chemical reactions. First, a bound transition state analog, which closely resembled the product of fatty acyl-CoA ester oxidation by medium chain acyl-CoA dehydrogenase (MCAD), was found to regulate the reaction by appropriately shifting its redox potential. In contrast, the substrate of methane monooxygenase (MMO) plays no role in activating the catalytic component of the enzyme; rather the binding of reductase in the presence of the regulatory component had a profound and unprecedented effect on the thermodynamics and kinetics of MMO. Our goal in the next grant period is to build on our discoveries in the following ways: l) Provide Raman spectroscopic evidence for the hypothesized polarization of substrate/product in the active site of MCAD. These spectroscopic studies will provide evidence for a proposed transition state and enable us to elucidate the mechanism on the molecular level. 2) Test the hypothesis that the electron transfer reaction between the RI and R2 components of ribonucleotide reductase (RNR) is likely gated by both complex formation between RI and R2 and substrate binding in the RI subunit with further control mediated by the binding of allosteric effectors. 3) By using Acyl-CoA Desaturase, establish a new global regulation mechanism for enzymes by providing evidence that regulation by regulatory proteins is observed in other systems besides MMO and RNR.
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