No description available.

No description available.

No description available.

No description available.

Many biologically important electron transfer reactions involve iron-sulfur centers and flavosemiquinones. Laser flash photolysis and stopped-flow spectrophotometry has been utilized to investigate factors influencing transient kinetic oxidation of free and protein-bound flavosemiquinones by various types of oxidants, but primarily high potential iron-sulfur proteins (HiPIP) and rubredoxin classes of Fe-S proteins. Studies of free flavosemiquinone analog oxidation by Fe-containing redox proteins and inorganic oxidants have found the reactivity of the anionic flavosemiquinone to be greater than that for neutral flavosemiquinone. The second order rate constants of oxidation of various flavosemiquinone analogs by either redox proteins or non-biological oxidants correlate with the difference in redox potential of the reactants according to the Marcus exponential equation. For the protein-flavin analog reactions, deviations from the theoretical Marcus curve are interpreted in terms of the effects of the different exocyclic substitutions on intrinsic anion semiquinone reactivity. Electrostatic effects on FMN and C. pasteurianum flavodoxin semiquinone oxidation by HiPIP can be quantitated yielding the rate constant at infinite ionic strength (k∞) and the charge product for reaction. The magnitude of the electrostatic effects are larger for flavodoxin semiquinone oxidation than for FMN semiquinone oxidation which is consistent with the larger electrostatic charge for flavodoxin. The k∞ values obtained from the electrostatic analysis for FMN semiquinone and flavodoxin semiquinone oxidation indicate a dependence on the redox potential difference between the reactants (ΔE). The ΔE effect is larger in magnitude for the flavodoxin reaction than for the FMN reaction. Deviations from the theoretical curves for the FMN and flavodoxin reactions suggest that factors other than redox potential and electrostatics, such as sterics, could be having an important influence on reactivity. The results suggest that electrostatics, redox potential, and perhaps sterics could have an important role in determining the biological specificity of protein/protein redox reactions. C. pasteurianum rubredoxin forms a 1:1 complex with spinach ferredoxin:NADP+- reductase (FNR) at low ionic strengths. The reduction of Rdₒₓ by lumiflavin semiquinone is unaltered in the complex. Evidence is presented for a rapid (2 x 10³s⁻¹) intracomplex electron transfer from FNR semiquinone to oxidized rubredoxin.

Thesis (Ph. D.)--Ohio State University, 2004. / Title from first page of PDF file. Document formatted into pages; contains xii, 153 p.; also includes graphics. Includes abstract and vita. Advisor: Richard P. Swenson, Dept. of Biochemistry. Includes bibliographical references (p. 144-153).

It was estimated that more than one hundred open reading frames in Pyrococcus furiosus and Thermotoga maritima could encode flavoproteins based on the results of motif search and comparison of genomic annotation to the experimentally characterized flavoproteins. However, only a few flavoproteins have been characterized from those anaerobic hyperthermophiles. It was found T. maritima and Thermotoga hypogea were able to grow in the presence of micromolar level of oxygen. As part of an oxygen removal system, the presence of NADH oxidase was detected in both microorganisms. In T. hypogea, NADH oxidase activity was constant regardless of the presence of oxygen, while in T. maritima it was increased in the presence of oxygen. The purified T. hypogea NADH oxidase was a flavin adenine dinucleotide (FAD)-containing homodimer with subunit molecular mass of 50 kDa. In addition to NADH oxidase activity, it also demonstrated activity of dihydrolipoamide dehydrogenase (DLDH), which is probably involved in glycine decarboxylation. The purified NADH oxidase from T. maritima was a heterodimeric protein of two subunits with molecular weight of 54 and 46 kDa, which were identified to be encoded by TM1432 and TM1433, respectively. Each subunit bore one FAD and the large subunit had one bacterioferritin-associated ferredoxin (BFD)-like [2Fe-2S]-center. Although the T. maritima NADH oxidase had very unusual oxygen sensitivity, the oxygen inactivated enzyme could be fully recovered by incubating with reducing reagents anaerobically. The NADH oxidases from both T. hypogea and T. maritima catalyzed the reduction of oxygen only to hydrogen peroxide. NADH-dependent peroxidase activities were detected in both T. maritima and T. hypogea, suggesting the presence of a multi-component oxygen detoxification system in Thermotoga species. In addition to its NADH oxidase activity, the enzyme from T. maritima exhibited FAD-linked glycerol-3-phosphate dehydrogenase (FAD-GPDH) activity. Along with the glycerol kinase, the FAD-GPDH took part in glycerol utilization in T. maritima. Ferredoxin NAD+ oxidoreductase (FNOR) activity was detected in T. maritima using an NADH:benzyl viologen oxidoreductase (BVOR) assay. The purified enzyme was a homodimeric FAD-containing protein with subunit molecular mass of 37 kDa. The purified enzyme was very active in catalyzing the reduction of BV and methyl viologen (MV) using either NADH or NADPH as electron donor and could indeed catalyze the reduction of NAD+ with the reduced ferredoxin from T. maritima. The purified enzyme was further identified to be encoded by TM0869 and annotated as thioredoxin reductase (TrxR). T. maritima TrxR could not use commercially available thioredoxin (Trx) from Spirulina, but the Trx purified from T. maritima. T. maritima Trx was identified to be encoded by TM0868 and annotated as glutaredoxin (Grx)-like protein, which showed both thioredoxin (Trx) and Grx activity. The purified T. maritima TrxR could catalyze the Trx-dependent reduction of both insulin and DTNB using NAD(P)H as electron donor. The identified Trx-TrxR system in T. maritima is the first one characterized in hyperthermophilic bacteria. T. hypogea has great potential in microbial hydrogen production. The key enzyme involved in this process, hydrogenase, has not been studied yet. The growth-dependent hydrogenase activity was detected in T. hypogea, from which a homotetrameric hydrogenase was purified. The purified T. hypogea hydrogenase did not contain any flavin prosthetic group as speculated, but [Fe-S]-centers. The hydrogenase could catalyze both BV and MV-dependent hydrogen oxidation and MV-dependent hydrogen evolution. Neither NAD(P)H nor NAD(P) could be used as electron carrier for this enzyme. T. hypogea hydrogenase could utilize ferredoxin as electron carrier for both production and oxidation of hydrogen, which suggests that the purified hydrogenase plays an important role in hydrogen metabolism of T. hypogea. It was concluded that flavoproteins can be involved at least in several very important cellular processes such as detoxification of oxygen, utilization of glycerol, redox regulation, and hydrogen metabolism in hyperthermophiles.

No description available.

published_or_final_version / abstract / toc / Botany / Doctoral / Doctor of Philosophy

Thesis (Ph. D.)--University of Delaware, 2007. / Principal faculty advisor: Colin Thorpe, Dept. of Chemistry & Biochemistry. Includes bibliographical references.