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Potential of mean force calculations were performed on the wild-type medium-chain acyl-CoA dehydrogenase (MCAD) and two of its mutant forms. The study demonstrates that significant insights into the catalytic mechanism can be obtained from simulation studies, and it is possible to design novel mechanistic probes for the enzyme with the help of the result.

The thermodynamic regulation is examined through direct characterization of the electrochemical properties of human short-chain acyl-CoA dehydrogenase (hSCAD) using spectroelectrochemical methodology. The result indicates that in hSCAD, substrate redox activation occurs that leads to a large enzyme midpoint potential shift and substrate binding appear to make a larger contribution than does product to thermodynamic modulation.

Short-chain acyl-CoA dehydrogenase (hSCAD) catalyzes the first matrix step in the mitochondrial beta-oxidation cycle with optimal activity toward butyryl- and hexanoyl-CoA. The result provides valuable insight into the mechanistic basis for dysfunction of the common variant hSCADs and demonstrates that mutations, regardless of their position in the protein structure, have a large impact on the redox properties of the enzyme.

Research has been conducted on the aromatic stacking interactions. The results from the redox studies indicate that both the phenylalanine and tyrosine engage in pi-sigma interactions with isoalloxazine flavin ring to stabilize anionic flavin hydroquinone formation.

Results point out that there is a selective polarization of 2,4-Hexadienoyl-coenzyme A active site upon binding to oxidized and reduced medium-chain acyl-CoA dehydrogenase from pig kidney. Data indicate that midpoint potential of the above complex is dependent on pH with a pKa around 9.2.

Medium-chain acyl-CoA dehydrogenase (MCADH) is the best studied member of a broad family that catalyzes the dehydrogenation of fatty acids. The reductive half-reaction, which proceeds via the abstraction of both a proton and a hydride ion, has been studied. It was found that substrate/product couple binding raises redox potential of MCADH by more than 100 mV, enabling unfavorable electron-transfer.

The quantitative thermodynamic data of the nitric oxide synthase (NOS) heme was described with the determination of the heme midpoint reduction potentials for the neuronal NOS and inducible NOS oxygenase domains. Results showed that the binding affinities of L-arginine and tetrahydrobiopterin increase by about 80-fold and 8-fold for the reduced heme iron form of the enzyme. These explain the contrasting reactivities between the two NOS isoforms regarding their different NADPH consumption rates and capacity to support heme iron reduction.

Research was conducted to examine the oxidase activity of the acyl-CoA dehydrogenases. A second chromophoric substrate of the medium chain enzyme was used and another representative of the acyl-CoA dehydrogenase family was tested. Results indicate that oxygen interacts not only with the free enzyme but also with several reduced enzyme species. These results underscore the complexity of an apparently simple reaction as the reoxidation of the acyl-CoA dehydrogenase by molecular oxygen.

The functional properties of four site-directed vegetative cell ferredoxin (Fd) mutants, C41S, C46S, C49S and C79S, and an X-ray crystallographic structure for C49S are presented. Each mutant has the ability to rapidly accept an electron from deazariboflavin semiquinone and to transfer an electron from its reduced form to oxidized ferredoxin:NADP+ reductase (FNR) although they are less reactive toward FNR and are less stable in solution than is wt Fd. Results suggest that the reactivity of the mutants is not correlated with altered reduction potentials.

Nonconservative mutations of the critical residues S47, F65, and E94, on the surface of ferredoxin have large parallel effects on the reduction potential and the electron-transfer reactivity of the (2Fe-2S) cluster. The reduction potential changes are not the principal factor governing electron transfer reactivity. The specific orientation of the proteins within the transient electron-transfer complex control the kinetic properties Anabaena Ferredoxin.

The regulatory effects of methane monooxygenase reductase (MMOR) on the redox potentials of methane monooxygenase hydroxylase (MMOH) and its complexes, the kinetics of the reaction cycle steps, and the product yield of catalysis were investigated. MMOR was found to be essential in MMOH's catalytic function. The effects of reductase component and component B on hydrolase component was shown to be important for the regulation of catalytic efficiency and rate in an oxygenase system.

The catalytic performance of several wild-type and mutant short-chain acyl-CoA dehydrogenases (SCAD) containing oxirane during the 1,3-polymerization between alpha,beta- and beta,gamma-enone thioesters was investigated. The results, which demonstrated the failure of the mutant flavoprotein to catalyze the gamma-H exchange/abstraction, proved SCAD-catalyzed allylic isomerization to be a one-base mechanism.

The potentials of the redox cofactors of enzymes CDP-6-deoxy-L-threo-D-glycero-4-hexulose-3-dehydrase (E1) and CDP-6-deoxy-delta(super 3,4)-glucoseen reductase (E3), which are involved in the C-3 deoxygenation process in the biosynthesis of ascarylose in Yersinia pseudotuberculosis V, were determined to examine the electron transfer performed. Findings indicated that the redox characteristics of this system can be controlled by pH and the electron transfer between the E3 redox centers can be prototropically regulated.

A study was conducted to characterize the electron transfer properties of the R2 protein of ribonucleotide reductase from Escherichia coli. The results show that ribonucleotide reductase R2 is a dimer comprised of two dinuclear iron centers with redox potential of -115 millivolts, and a tyrosyl radical with redox potential of 1,000 plus or minus 100 millivolts.

A study was conducted on the influence of transition-state analogues on the redox potential of medium-chain fatty acyl-CoA dehydrogenase (MCAD). Potentiomeric and coulometric measurements were taken and analyzed. Results indicated that the formation of a product-like transition state induces the positive shifting of MCAD, subsequently providing the thermodynamic energy for electron transfer.

X-ray studies help determine the structure of the complex formed between butyryl-CoA dehydrogenase (BCAD) from Megasphaera elsdenii and acetoacetyl-CoA. The molecular replacement method uses the atomic coordinates of pig liver medium chain acyl-CoA dehydrogenase (MCAD) to determine the structure of BCAD. Substrate specificities and enzymatic differences between BCAD and MCAD are discussed with respect to differences in the side chains of the helices making the binding site.

Product binding regulates the thermodynamic characteristics of an active-domain of a Megasphaera elsdenii short chain acyl-CoA dehydrogenase (SCAD). A resonance structure of the product is identical to an intermediate in the catalytic process which plays a significant role in enabling the transfer of isopotential electrons to the enzyme from the substrate. The reduction potential of Glu(super 367)Gln SCAD is not significantly influenced by butyryl-CoA binding.

A combination of electrochemical, EPR and Mossbauer spectroscopic studies conducted on reductive reactions of methane monooxygenase hydroxylase (MMOH) in the presence of MMO-reductase, MMOB and propylene, yields implicit potential values for each of MMOH's redox states. MMOH from Methylosinus (M) trichosporium OB3b differs in the two primary redox values of MMOH obtained from the enzyme M. capsulatus. Redox-dependent structural variations in MMOH relate to MMOH affinity for complex formation.

The thermodynamic characteristics of medium-chain acyl-CoA dehydrogenase (MCAD) are controlled by a substrate or a product whose important conformational workings are recognized. MCAD is used to complex two groups of substrate analogues, acetyl-CoA and thioether-CoA, which influence the redox characteristics of MCAD. A comparison between the redox characteristics of complexed and uncomplexed MCAD is possible by examining the pH reliance of the redox potency of uncomplexed MCAD. This also allows the measurement of the dissociation steady factors of the analogues to the three redox states of MCAD.

Megasphaera elsdenii reveals a short-chain acyl-CoA dehydrogenase (SCAD), which exhibits a molecular mechanism. The functioning is controlled by cloning and ordering the gene coding for SCAD. The suggested functioning needs an amino acid residue that is recognized by site-directed mutagenesis. Identification of the amino acid order of the 50 N-terminal residues of SCAD is required to clone the gene. Two groups of varying oligonucleotide primers are prepared by the order of the data, and a 120-bp specific probe from Megasphaera elsdenii DNA is produced by a polymerase chain reaction process.

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.

The role of the oxyanion hole in Mechanistic studies on the medium-chain enzyme (MCAD) catalysis is been elaborated using a combination of kinetic, Raman spectroscopy, and redox combination of measurements. Numerous studies and their respective results concerned with the same are mentioned.

A study was conducted to show that tyrosyl radical (Y122) formation supports two functionally distinct diiron(II) clusters, one that becomes the (Y122, FE(super III)-O-Fe(super III)) site and another that promotes the needed extra electron and forms the (Y122, Fe(super III)-O-Fe(super III)) site. The chemical reaction of the R2 component was represented as a function of time. Results indicated a basis for a strategy ascribing distinct roles for the diiron clusters.

Cyclobutanemethanol is the starting material for cyclobutaneacetyl-CoA (CBA-CoA) and the product is unique due to its duality in activity. CBA-CoA is a substrate for medium-chain acyl-CoA dehydrogenase (MCAD) and is an inhibitor of long-chain acyl-CoA dehydrogenase (LCAD). Reactions with MCAD involve C-beta-H release and reactions with short-chain AD involve cleavage of C-beta-C-gamma linkage. The Janus-faced nature of CBA-CoA is responsible for the selective bond cleavage and the active sites of MCAD and SCAD induce different steric and electronic demands.