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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.

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.

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.

A series of selective labelling procedures conducted on the enzyme dihydrofolate reductase (DHFR) of Lactobacillus casei revealed that the pKa value of the aspartic acid residue of the enzyme does not exceed the 4.8 level. The occurrence of site-directed mutagenesis in the resonance of Asp 26 further showed that the active site of the DHFR residue exists in the ionized form. It was also established that the polarization of dihydrofolate may be initiated in the presence of NADPH.

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.

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.

A kinetic data for a variant of ascorbate peroxidase (W41A) which reacts slowly with tert-butyl hydroperoxide without forming the usual peroxidase Compound I intermediate is used to explain the reaction mechanism and function of heme peroxidase enzyme. The findings for heme oxygenase reaction mechanism provides insight into the formation of product in which the heme is cleaved at the [alpha]-meso position, analogous to the heme oxygenase mechanism.

The mechanisms involved in the formation of a covalent link between the distal tryptophan (Trp51) and the heme in cytochrome c peroxidase (CcP) are discussed. The properties of the radical thus formed are shown to be highly dependent on the structure of the compound I intermediate.

The activity of 1-Me-L-Trp with three different heme dioxygenases and their site-directed variants is examined. The studies have shown that 1-Me-L-Trp is a substrate for the heme dioxygenase enzymes, the deprotonation of the indole [N.sub.1] is not essential for catalysis and an alternative reaction mechanism is described.

The closely related cytochrome c peroxidase enzyme has duplicated the substrate binding properties of ascorbate peroxidase by introducing relatively modest structural changes at Tyr36 and Asn184. The studies have shown that the combination of efficient substrate binding along with the formation of a suitably stable phorphyrin [pi]-cation radical is the key to rapid substrate oxidation at the [gamma]-heme edge and that hydrogen bond has played a vital role.

The role of the conserved active site Ser167 residue in human indoleamine 2,3-dioxygenase (IDO) (S167A and S167H variants), which is replaced with a histidine in other mammalian and bacterial tryptophan 2,3-dioxygenase (TDO) enzymes, is described. The results have disclosed significant differences in reactivity between the IDO and TDO enzymes.

A bacterial expression system is described for human tryptophan 2,3-dioxygenase (rhTDO) along with spectroscopic, kinetic and redox analyses. The studies have shown that the rhRDO is catalytically active in the ferric form and significant differences are present across the heme dioxygenase family.

The data from X-ray crystallographic studies are used to demonstrate the conformational rearrangement due to reduction of the heme iron which leads to dissociation of His42 from the iron in the ferrous form of W41A.

The anomalous negative fluorescence anisotropy observed in yellow fluorescent protein (YFP) is discussed. The findings show that the negative anisotropy is a result of the unidirectional Forster resonance energy transfer (FRET) in its dimers.