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A complex formation between the methionine synthase (MS) activation domain and methionine synthase reductase (MSR) is investigated. It shows that the MS activation domain interacts directly with the FMN binding domain of MSR.

The heme domain of the Bacillus megaterium fatty acid hydroxylase flavocytochrome P450 BM3 is rapidly reduced when NAD(P)H is photoexcited at 355 nm using an Nd:YAG laser. The rate of formation of the iron-carbon monoxide complex increases resulting into a rapid electron transfer to P450 enzymes.

[alpha]-Secondary kinetic isotope effects (2 degree KIEs) are used in conjunction with primary 1 degree KIE to investigate the mechanism of environmentally coupled hydrogen tunneling in the reductive half-reactions of two homologous flavoenzymes, morphine reductase and pentaerythritol tetranitrate reductase. The analysis has stressed on the benefit of combining studies of 1 degree and 2 degree KIEs to describe the pre-organization and local geometries within the context of contemporary environmentally coupled frameworks for H-tunneling.

Maize polyamine oxidase (MPAO), a flavin adenine dinucleotide (FAD)-dependent enzyme that catalyses the oxidation of spermine and spermidine at the secondary amino groups is studied. It indicates that oxidized FAD is the prominent form during steady-state turnover, in the enzyme-monitored turnover method, consistent with reductive half-reaction being rate-limiting.

The kinetic mechanism of dimethylglycine oxidase reveals typical flavoprotein 'oxidase' properties in that the formation of a reduced flavin-iminium charge-transfer intermediate in the reductive half-reaction is similar to those mediated by other oxidases. Data indicate that decay of the intermediate is rate-limiting for the enzyme's steady-state reactions.

Results demonstrate that replacement of Trp-676 with His-676 in the cytochrome P450 reductase results in about 2-fold reduction in the rate of electron transfer in both 'forward' and 'reverse' directions. Also, the internal electron transfer in two- and three-electron reduced cytochrome P450 reductase is slow.

Research has been conducted on the human cytochrome P450 reductase. The redox center reduction in this reductase has been investigated via rapid-mixing stopped-flow spectroscopy and the results are discussed.

Research reveals that both W676H and W676A, mutant forms of human cytochrome P450 reductases, mediate nicotinamide exchange during the reductive half-reaction of the reductase. The mutant enzymes contain histidine and alanine at the position of Trp-676 of the wild cytochrome P450 reductase.

The fact that matter can be described as both a particle and a wave means that hydrogen nuclei could tunnel through a potential energy barrier occurring at the catalytic site. Kinetic behavior consistent with quantum tunneling has been found in several enzymes.

Electron transfer between the 4Fe-4S center of trimethylamine dehydrogenase and electron transferring flavoprotein involves the Tyr-442 residue but not the Val-344 residue. Mutation of Tyr-442 reduces electron transfer but mutation of Val-344 does not.

Recombinant wild-type and a C30A mutant of trimethylamine dehydrogenase (TMADH) from Methylophilus methylotrophus (sp. W(sub.3)A(sub.1)) have been characterized from the perspectives of structure and biochemistry. Combined crystallographic and NMR studies show inorganic phosphate in the FMN binding site in the deflavo fraction of both recombinant wild-type and C30A proteins. Major differences in kinetic behavior are seen in solution studies. Computational studies show contribution made by the S(gamma) atom of Cys-30 to substrate binding and a role for Cys-30 in the optimal geometrical alignment of substrate with the 6-S-cysteinyl FMN in the enzyme active site.

Research has been conducted on the heterotetrameric sarcosine oxidase reaction of Arthrobactor sp. 1-IN. The use of stopped-flow spectroscopy is described.

Redox cycles in trimethylamine dehydrogenase (TMADH) and the mechanism of substrate inhibition are discussed. The steady-state reaction of TMADH with the artificial electron acceptor ferricenium hexafluorophosphate (Fe(super.+)) has been studied. Partitioning between the 0/2 and 1/3 redox cycles depends on both trimethylamine (TMA) and electron-acceptor concentration. Reduction of Fe(super.plus) takes place via the 4Fe-4S center of TMADH, not the 6-S-cysteinyl flavin mononucleotide.

The enzymatic breakage of the substrate C-H bond by Methylophilus methyltropus methylamine dehydrogenase has been investigated. The results of stopped flow spectroscopic studies showed that the rate of reduction of the tryptophan tryptophylquinone cofactor has a large kinetic isotope effect which is independent of temperature. Analysis of the temperature dependence of C-H bond breakage showed that extreme quantum tunneling was responsible for the transfer of the hydrogen nucleus.

A study was conducted to analyze the reaction of morphinone reductase (MR) from Pseudomonas putida M10 with NADH and the oxidizing substrate codeinone. An Applied Photophysics SF.17MV stopped-flow spectrophotometer was utilized to carry out rapid reaction kinetic experiments while time-dependent reductions of MR with NADH were determined by multiple wavelength stopped-flow spectroscopy. Results showed that the reaction pathway from the oxidative half-reaction of MR is not optimal.

Analysis of the samples of the native and recombinant wild-type trimethylamine dehydrogenases provided the description of the properties of of a trimethylamine dehydrogenase aromatic residue mutant, W355L. Data indicated that the rationale for the existence of 8alpha-methyl and C6-linked covalent flavoproteins is the possible formation of an electrophilic iminoquinone methide in the hydroxylation of flavin.

The factors affecting alkylammonium ion specificity were investigated using bacterial trimethylamine dehydrogenase (TMADH) as the experimental molecular model system. A closely related enzyme, dimethylamine dehydrogenase and mutants of the two enzymes were also included in the assays to provide bases for comparison of enzyme activities. The results of the kinetic studies revealed that a selective modification of the substrate-binding site in TMADH is responsible for the change in substrate specificity exhibited by the mutant enzymes.

The kinetics of electron transfer in wild-type and mutant trimethylamine dehydrogenase*electron transferring flavoprotein (TMADH*ETF) electron transfer complexes were studied to determine the surface residues of TMADH that interact with ETF and potential electron transfer. Three mutant enzymes in which residue Tyr-442 is exchanged for a phenylalanine, leucine or glycine residue were isolated and characterized. Findings showed that the concave region of TMADH forms part of the interaction site for ETF.

Stopped-flow spectroscopy helps determine the rate constant for the reoxidation of substrate-reduced trimethylamine dehydrogenase by ferricenium hexafluorophosphate, an artifical electron acceptor. Examination of the dependence of the second electron transfer on temperature helps determine the reorganizational energy and the electronic coupling matrix element. Electron transfer from the enzyme to ferricenium ions proceeds via Tyr-442. The distance of electronic tunneling pathway is determined. The surface of the enzyme has the binding site for the physiological electron acceptor.

A study of the oxidative and reductive half-reactions of glutathione reductase from Escherichia coli using the wild type enzyme and a H439A altered form reveals that the transfer of electrons from reduced flavin to the disulfide in both half-reactions is affected by the substitutions. FAD reduction manifests as a decrease at 460 nm and an increase at 670 nm when the rate of reduction remains at 250 per sec. The reductive half-reaction limiting rate in the wild-type enzyme is less than that of the oxidative half-reaction and is independent of the NADPH concentration.

The systematic conversion of the NAD-dependent enzyme, Escherichia coli dihydrolipoamide dehydrogenase, into one that rejects NAD but is fully active with NADP is presented. In addition, the ability of the mutated enzyme to assemble into a fully active pyruvated dyhydrogenase multicomplex enzyme complex was examined. The specificity of the assembled product was also assessed. It was found to catalyze the oxidative decarboxylation of pyruvate to yield acetyl coenzyme-A.

The procedure devised to add penta-arginine stretch of amino acids to the N-terminus of Escherichia coli glutathione was applied to the isolation and characterization of heterodimers of glutathione reductase in the study of two active-site mutants, H439Q and Y177G. The introduction of the C47S mutation removed the essential charge-transfer cysteine residue and allowed the selective inactivation of one active site in the hybrid. This study is a demonstration of a method of generating separable enzyme hybrids that can be used in investigating catalytic mechanisms and structure assembly.

A procedure for selectively tagging subunits of Escherichia coli glutathione reductase by engineering benign alterations to the surface charge was devised. This technique was employed to re-examine glutathione dissociation induced by reduced nicotinamide adenine dinucleotide phosphate. The extensions of this technology as applied to detecting hybrid formation and the causative subunit dissociation and exchange is discussed.

The various effects of the donor-acceptor distance and the isotope effects on the force constant exhibited during barrier compression in the H-tunneling reactions are analyzed. The force constant is shown to highly increase with a decrease in the donor-acceptor relationship of the system.

Continuous wave photolysis magnetic field effect (MFE) data has disclosed the ethanolamine ammonia lyase (EAL) active site to be an ideal microreactor for observing enhanced magnetic field sensitivity in the adenosylcobalamin radical pair. The results have shown that rapid radical quenching, through the coupling of homolysis and hydrogen abstraction steps, and subsequent radical pair stabilization has contributed to the rate acceleration of Co-C bond homolysis in adenosylcobalamin-dependent enzymes.

The single hydride transfer from both NADPH and NADH to FMN in the reductive half-reaction of pentaerythritol tetranitrate reductase (PETNR) is examined. The studies have ruled out differences in rate due to variation in driving force and the donor-acceptor distance, which has shown that it is the promoting vibration with NADPH that is the origin of the increased observed rate.

The catalysis of trans addition of hydrogen across the C17-C18 double bond of protochlorophyllide during chlorophyll biosynthesis is carried out by the light-driven enzyme protochlorophyllide oxidoreductase. The ternary enzyme-substrate formed during the reaction is investigated for the reduced activity of the enzyme.

A theoretical treatment of the combined effect of temperature and pressure on environmentally coupled H tunneling reactions is described. The pressure dependence of the kinetic isotope effect (KIE) for an H-transfer reaction has provided a method of determining the contribution of nuclear quantum mechanical (NQM) effects to the reaction, with a pressure-dependent KIE suggestive of NQM tunneling reaction.

The variational transition-state theory calculations are employed to study the hydride transfer taking place from nicotinamide adenine dinucleotide (NADH) to flavin mononucleotide (FMN) in morphinone reductase. The transfer is shown to be biologically important and is completely dominated by the deep tunneling phenomenon.

The significant role played by Arg-[alpha]237 in complex assembly, electron transfer, and imprinting of electron transferring flavoprotein (ETF) was examined through the isolation and study of several mutant ETFs. The results confirmed the importance of Arg-[alpha]237 to control the thermodynamics of electron transfer, the dynamics of ETF, and the protection of reducing equivalents following disassembly of the trimethylamine dehydrogenase (TMADH)-2ETF complex.

Kinetic isotope effects (KIEs) are combined with structure-reactivity studies to show that para-substituted benzylamines are good reactivity probes of tryptophan tryptophylquinone (TTQ) mechanism with the isolated TTQ-containing subunit of aromatic amine dehydrogenase (AADH). The para-substituted benzylamines are good reactivity probes of TTQ mechanism with the isolated small subunit, which is attributed to the absence of structural rearrangement prior to H-transfer that has limited the rate of TTQ reduction by para-substituted benzylamines in native enzyme.

The article shows that when it is rate-limiting in presteady-state kinetic studies with ethanolamine ammonia lyase (EAL), the net forward rate of C-Co bond homolysis is insensitive to the application of external magnetic fields in the presence of substrate. The studies have strong implications for the mechanism of radical pair (RP) stabilization and the unprecedented catalytic power of this class of cobalamin-dependent enzymes.

Stopped-flow spectroscopy has demonstrated that reduction of the FMN cofactor by NADH in N189A morphinone reductase (MR) in multiphasic, thus identifying four different reactive configurations of the MR-NADH complex. The analysis has helped in deriving the mechanistic information regarding multiple reactive configurations of the same enzyme-coenzyme complex by using stopped-flow methods.

Report is presented on the crystal structure of the methionine synthase reductase (MSR) flavin mononucleotide/nicotinamide adenine dinucleotide phosphate FAD/NADP(H)-binding module. The crystallographic structure of the ferrodoxin-NADP(H) reductase (FNR) module of MSR provides a framework to understand the catalytic properties of MSR.

The quinoprotein mechanism for aromatic amine dehydrogenase was studied by structure-activity correlations earlier. However, it is shown by combining the use of kinetic isotope effects (KIEs) and crystallographic analysis that para-substituted benzylamines are poor reactivity probes for TTQ-dependent aromatic amine dehydrogenase (AADH).

Multiple solution-state methods are used to investigate the nature and control of electron transfer in the context of the proposed 'domain shuffle hypothesis' for intraprotein electron transfer inferred from the crystal structure of the nitric oxide synthase reductase domain. Solution data is provided that are consistent with the proposed domain shuffle hypothesis for electron transfer neuronal nitric oxide synthase (nNOS), inferred from the crystallographic structure of nNOS reductase.

Mutant forms of DMGO were isolated to determine whether residues His-225 and Tyr-259 are involved in abstraction of a proton from the substrate amine group of dimethlyglycine prior to C-H bond breakage and FAD reduction. It was found that both mutant enzymes retain the ability to oxidize substrate and His-225 is likely to modulate the acid-base properties of Tyr-259.

An attempt is made to identify different conformational states in human methionine synthase reductase (MSR) and their role in electron transfer. With stopped-flow kinetic studies of the related enzymes cytochrome P450 reductase and nitric oxide synthase, comparisons were made.

Results show that flavin reduction/oxidation kinetics can be regulated by the coenzyme occupying the noncatalytic binding site that restricts coenzyme release from the catalytic site. Data indicate that cysteine plays a role in hydride transfer in the diflavin reductase family of enztmes and that Cys999 participates in the binding of NADP at the FAD domain.

Results demonstrate that the unconventional trend of kinetic isotope effect on the reduction of PQQ-dependent methanol dehydrogenase arises because of the differential binding affinity of the enzyme for methanol and deuterated methanol. Furthemore, the competitive binding of both species of methanol to binding sites of ammonium and phenazine ethosulfate also contributes toward this anamoly.

Research indicates that the mechanism of electron transfer in human methionine synthase reductase is similar to those in diflavin oxidoreductases in that upon hydride transfer from NADPH to the oxidized FAD cofactor, the electrons disproportinate between the flavins.

A detailed reinvestigation of a reported magnetic field effect (MFE), in the haem-dependent enzyme horseradish peroxide (HRP) by the use of a stooped-flow device adapted to make high-precision single-wavelength measurements in the presence of a static magnetic field is presented. The results demonstrate the absence of MFE in the reductive half-reaction of HRP-catalyzed cycle and identify problems with the analysis of kinetic data in the original study.

A study was undertaken to show the electron transfer (eT) reactions of the rubredoxin reductase (RR)-rubredoxin eT complex in the Pseudomonas oleovorans through kinetic and thermodynamic analysis. The protein-protein complex, in conjunction with the membrane-bound omega-hydroxylase, were responsible for the hydroxylation of hydrocarbons. Results revealed that the reductive half-reaction of RR transpired through the reduction of the oxidized enzyme to an enzyme-NAD+ charge transfer segment. The iron-binding domains of rubredoxin, combined with RR, formed the functional eT complexes.

We demonstrate that photoexcitation of NAD(P)H at 355 nm using a Nd:YAG laser leads to rapid reduction of the heme domain of the Bacillus megaterium fatty acid hydroxylase flavocytochrome P450 BM3. An aqueous electron derived from photoexcited NAD(P)H is rapidly transferred to the heme domain, enabling the formation of a carbon monoxy complex of the ferrous P450 (FeII-CO) on the microsecond time scale. Using this approach we have determined the limiting rate constant (1770 s-1 for substrate-free heme domain) for formation of the FeII-CO complex. We find no dependence of the observed rate of FeII-CO complex formation on NAD(P)H concentration but demonstrate a hyperbolic dependence on carbon monoxide concentration. The apparent dissociation constant for the complex of carbon monoxide bound noncovalently to the ferric form of the BM3 heme domain (and with NADH as reductant) is 323 μM. Binding of a P450 substrate (N-palmitoylglycine) weakened the complex between carbon monoxide and the ferric BM3 heme domain (Kd increased to 1404 μM) but enhanced the rate of formation of the FeII-CO complex (3036 s-1 for substrate-free heme domain). This study demonstrates the applicability of NAD(P)H photoexcitation as a method for rapid electron delivery to P450 enzymes and provides a new route to probing the P450 catalytic cycle and its transient intermediates. [ABSTRACT FROM AUTHOR]