Flavoprotein

Abstract: NADH:ubiquinone oxidoreductase (complex I) is a major source of reactive oxygen species in mitochondria and a significant contributor to cellular oxidative stress. Here, we describe the kinetic and molecular mechanism of superoxide production by complex I isolated from bovine heart mitochondria and confirm that it produces predominantly superoxide, not hydrogen peroxide. Redox titrations and electron paramagnetic resonance spectroscopy exclude the iron-sulfur clusters and flavin radical as the source of superoxide, and, in the absence of a proton motive force, superoxide formation is not enhanced during turnover. Therefore, superoxide is formed by the transfer of one electron from fully reduced flavin to O2. The resulting flavin radical is unstable, so the remaining electron is probably redistributed to the iron-sulfur centers. The rate of superoxide production is determined by a bimolecular reaction between O2 and reduced flavin in an empty active site. The proportion of the flavin that is thus competent for reaction is set by a preequilibrium, determined by the dissociation constants of NADH and NAD+, and the reduction potentials of the flavin and NAD+. Consequently, the ratio and concentrations of NADH and NAD+ determine the rate of superoxide formation. This result clearly links our mechanism for the isolated enzyme to studies on intact mitochondria, in which superoxide production is enhanced when the NAD+ pool is reduced. Therefore, our mechanism forms a foundation for formulating causative connections between complex I defects and pathological effects.

Abstract: The NPH1 gene of Arabidopsis thaliana encodes a 120-kilodalton serine-threonine protein kinase hypothesized to function as a photoreceptor for phototropism. When expressed in insect cells, the NPH1 protein is phosphorylated in response to blue light irradiation. The biochemical and photochemical properties of the photosensitive protein reflect those of the native protein in microsomal membranes. Recombinant NPH1 noncovalently binds flavin mononucleotide, a likely chromophore for light-dependent autophosphorylation. The fluorescence excitation spectrum of the recombinant protein is similar to the action spectrum for phototropism, consistent with the conclusion that NPH1 is an autophosphorylating flavoprotein photoreceptor mediating phototropic responses in higher plants.

Abstract: Publisher Summary This chapter discusses the preparation and properties of microsomal TPNH-cytochrome c reductase from pig liver. It is purified from microsomes both as a partially purified microsomal subparticle, and with lipase treatment and fractionation, as a soluble flavoprotein essentially homogeneous in the ultracentrifuge. The assay depends upon measurement of the rate of cytochrome c reduction at 550 mμ. Cytochrome c reductase activity in microsomes is associated with marked TPNH-neotetrazolium diaphorase activity. This activity is disproportionately lost upon lipase treatmeat of microsomes, and its loss may serve as an indication of loss of an intermediate cofactor in the intact microsome, or of loss of a specific environmental or configurational state of the enzyme. The steps of purification procedure described are: preparation of lipase, preparation of microsomes, lipase solubilization, pH precipitation, ammonium sulfate fractionation, column chromatography on hydroxylapatite, and calcium phosphate gel concentration. The prosthetic group of TPNH-cytochrome c reductase enzyme is flavin adenine dinucleotide, but the apoenzyme can be reactivated by FMN and FAD. The enzyme is specific for TPNH, but is relatively nonspecific for electron acceptor. TPNH-cytochrome c reductase has a pH optimum of 8.2, which appears to be independent of the buffer used. TPNH-cytochrome c reductase is markedly stimulated by low levels of p-chloromercuribenzoate; maximal activation is reached at 2 moles of PCMB per mole of flavin, and higher concentrations inhibit.