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A study attempts to determine the rate-determining step in the steady-state turnover of wild-type glutaryl-CoA dehydrogenase (GCD) by studying the deuterium kinetic isotope effects and transient state kinetics. Based on the transit times of the individual reactions occurring in the active site of GCD, it was inferred that the significant rate-determining step is the release of crotonyl-CoA product because the chemical steps and reoxidation of reduced FAD are much faster than the turnover of the wild-type GCD.

The study is conducted to determine the protonation of crotonyl-CoA by glutaryl-CoA dehydrogenase. The result states that the water involvement in catalysis by glutaryl-CoA dehydrogenase is previously unrecognized and is in conflict with a classically held intramolecular 1,3-prototropic shift for protonation of crotonyl-CoA dienolate.

The three-dimensional structures of human glutaryl-CoA dehydrogenase (GCD) are reported in its uncomplexed form and in complex with 4-nitrobutyryl-CoA. The plausible mechanism for dehydrogenation and decarboxylation are predicted based on the structural bases.

Results show that the oxidase/dehydrogenase ratio of human glutaryl-CoA dehydrogenase is about 75-fold higher with 4-nitrobutyryl-CoA than with that of glutaryl CoA substrate. Data indicate that the product of the enzymatic oxidation is 4-nitronate-but-2-enoyl-CoA. The 4-nitrobutyryl-CoA is a poor substrate.

The wild-type glutaryl-CoA dehydrogenase and its mutant forms decarboxylate glutaconyl-CoA without flavin involvement in the oxidation-reduction reactions. Spectral analysis of the Glu370Gln mutant shows a 725 nanometer absorbing species that exhibits first-order kinetics, indicating that it may be the delocalized crotonyl-CoA anion.

Results demonstrate that the oxidation-reduction characteristics of the human glutaryl-CoA dehydrogenase are similar to acyl-CoA dehydrogenases from other sources. Steady-state kinetics of substrate binding reveal proton abstraction property of the enzyme. Mutants exhibiting altered enzyme show reduced rate of substrate deprotonation.

The role of the T244 mutation, introduced into the alpha subunit of Paracoccus denitrificans transfer flavoprotein (ETF), on its kinetic and redox properties has been studied. Results indicate that the mutation had no significant effect on the global structure of ETF, based on its visible and near-ultraviolet absorption and circular dichroism spectra. Tyrosine fluorescence in the mutant protein remains constant even if two tyrosine residues are near the site of the mutation. These findings indicate that changes in the structure of EFT are minor and localized.

E261 is the catalytic base identified in human long chain acyl-CoA dehydrogenase by molecular models and mutagenesis methods. Atomic coordinates are used for constructing the molecular model. The E376 catalytic residue is not conserved in the dehydrogenase drain. The E261 base extracts the acyl-CoA substrate alpha-proton in the dehydrogenase catalytic pathway.

The formal potential values for every electron transfer to electron transfer flavoprotein-ubiquinone oxidoreductase (ETF-QO) were studied to allow the characterization of electron transport thermodynamics from acyl-CoA thioesters to the ubiquinone pool within the mitochondria. Results reveal that ETF-QO has a (4Fe-4S)(super 2+, 1+) cluster and FAD prosthetic group. Visible-spectroelectro-chemical titration found that reduction occurred almost at the same time in both redox centers. At pH values of 7.5 and at a temperature of 4 degrees celsius, formal potential values for electron transfer to ETF-QO were determined at E1' = +0. 028 V and E2' = -0.006 V to the FAD and E'= +0.047 V for the (4Fe-4S)(super 2+, 1+) cluster.

Double electron-electron resonance (DEER) measurements between a spin label and an enzymatically reduced flavin adenine dinucleotide (FAD) cofactor in Paracoccus denitrificans ETF which gave two distributions of distances, a major component centered at 4.2 [plusmn] 0.1 nm and a minor component centered at 5.1 [plusmn] 0.2 nm is reported. The observation of a second distribution suggested that human mitochondrial electron transfer flavoprotein (ETF), in the absence of substrate adopted some conformations that are intermediate between the predominant free and substrate-bound states.

Mutations are introduced to modulate the flavin adenine dinucleotide (FAD) redox potentials and examine its importance for electron transfer flavoprotein-ubiquinone oxidoreductase (ETF-QO) function. The studies have shown that the FAD is involved in electron transfer to ubiquinone but not in electron transfer from ETF to ETF-QO and hence iron-sulfur cluster is the immediate acceptor from ETF.

Mutations are introduced to eliminate the hydrogen bonds to cysteine [S.sub.[gamma]] atoms of the [4Fe-4S] cluster and the impact of the hydrogen bonds on redox potentials and activity is examined. The decrease in steady-state ubiquinone reductase activity has shown that reduction of the iron-sulfur cluster is necessary for activity and there is no detectable effect of the mutations on the flavin midpoint potentials.

The impact of a Glu370Asp mutation in glutaryl-CoA dehydrogenase on the proton transfer to dienolate intermediate is discussed.

The electron transfer flavoprotein (ETF) from Paracoccus denitrificans is compared to human ETF in terms of structure. The overall fold of the ETF from P. denitrificans is similar to that of the human enzyme, except for a single loop region. As in humans, ETF from P. denitrificans has three domains, two at the alpha-subunit and a third at the beta-subunit. Using sequence and structures of human and P. denitrificans ETF, a comprehensive sequence alignment is constructed for the ETF family. Analysis indicates that the immediate flavin environment helps preserve flavin chemistry.