Optical spectra and electronic structure of flavine mononucleotide in flavodoxin crystals.

The polarized single-crystal absorption spectra of the oxidized and semiquinone forms of flavodoxin from Clostridium MP have been measured with a double beam recording microspectrophotometer. The spectra establish that the radical species in the crystal is the neutral (blue) falvine semiquinone. Combination of the spectra reported here with polarization data from previous fluorescence and stretched-film studies provides transition moment directions for the first two phi-phi transitions of the oxidized form. Predictions of molecular orbital theory are in good agreement with these experimental directions. The crystal spectra of the semiquinone indicate that the two lowest frequency transitions have the same detailed orbital origin as the corresponding transitions of the oxidized form; in the semiquinone these transitions appear at lower frequency, are closer together, and, as predicted from detailed considerations of transition probabilities, exhibit approximately half the absorption intensity. Our hypothesis of a common orbital origin suggests that semiquinone formation takes place by the addition of an electron to the lowest empty phi orbital of the oxidized form without any gross electronic rearrangement.     

The kinetics of flavine oxidation--reduction. I. Dismutation in nonaqueous solvent.

The dismutation reactions of flavines in dimethylformanide have been investigated using the stopped-flow technique under anaerobic conditions. The ionization constants of fully reduced and oxidized tetraacetylriboflavine were measured spectrophotometrically in buffered dimethylformanide. The dismutation equilibrium of the flavine as a function of pH in dimethylformanide was roughly comparable to that reported in water and allowed the estimation of the pKa value of the flavosemiquinone. The dismutation kinetics of tetraacetylriboflavine in unbuffered dimethylformanide were investigated using the fully oxidized and reduced flavines in their neutral form at constant produce of concentrations and varying the reduction degree. The kinetics at very low reduction ratios (less than3%) were triphasic. The kinetic analysis of the initial and simultaneous formation of the anionic and neutral radicals revealed a second-order reaction. The electron transfer between the oxidized and reduced flavines was not directly coupled with prton exchange. The multiphasic time course of the reaction proceeded primarily from differences in the intrinsic rates of the direct and mixed backward dismutation reactions of the two radical species, and finally from a change in the equilibrium conditions resulting from the accumulation of anionic flavohydroquinone. An acidic-basic negative catalytic effect from the neutral flavohydroquinone appeared progressively as the reduction degree was increased. It was complete at reduction ratios higher than 30%, i.e. under conditions where the radical anion could not be observed at any reaction time. Acids with a pKa value lower than the second one of the flavosemiquinone exhibited a similar catalytic effect. These acidic-basic catalytic effects are associated with changes in the ionic state of labile intermediate dimers formed in the forward as well as in the backward direactions of the dismutation reaction. Such a transient complex revealed by the kinetic analysis could be observed directly by absorption spectroscopy in alkaline-buffered dimethylformanide. Its spectral characteristics, as well as the kinetic effects induced by substitution of the benzenoid part of the flavine, can hardly be taken into account by a quinhydrone-like structure for the intermediate dimers at any pH value. The experimental results favored a more specific interaction, possibly of covalent character, involving the benzenoid part of the isoalloxazine ring.     

The kinetics of flavine oxidation-reduction. II. Metal ion interactions.

The oxidation-reduction reactions of tetraacetylriboflavine in the presence of various metal ions in dimethylformamide have been investigated using the stopped-flow technique under anaerobic conditions. Dismutation kinetics in the presence of redox-inactive dissociated divalent metal ions such as Cd2+, Zn2+, and Fe2+ are typically triphasic. Metal ions act primarily upon an intermediate flavine dimer formed by fast association of flavoquinone and flavohydroquinone, resulting in a parallel formation and neutral and chelated radicals. A competition between metal ions and proton donors, e.g. the neutral flavohydroquinone (FredH3), is observed at the level of this intermediate complex. Small spectral changes occur secondarily as an ill-defined intermediate phase which could correspond to the reorganization of the solvation of radical chelate. The neutral radical is finally chelated at a much slower rate, the yield of total radical formation remaining almost unchanged during this kinetic phase. The oxidation of flavohydroquinone by ferric ions, either dissociated or strongly coordinated within a porphyrin, is complete and proceeds through biphasic kinetics. The first phase (Fred leads to F) is much faster than the second one (F leads to Fox). Dismutation resulting from the transient accumulation of neutral flavosemiquinone competes with the direct oxidation with ferric ions for the completion of the second oxidation step. The relative rate of dismutation is essentially limited by acidic-basic reactions in the absence of an excess of ferrous ion. The kinetic analysis of the direct oxidation reactions favors an outer-sphere mechanism for the electron transfer to the ferric ion, either free or strongly coordinated. The formation of a ferrous radical chelate can result from the dismutation reactions only when the amount of ferric ion initially present is not sufficient for complete oxidation.     

The modification of the cytotoxic effect of leucocidin by N-ethylmaleimide, flavine mononucleotide and menadione

1. The movement of the cytoplasmic granules in the leucocidin-treated leucocyte is prevented in the presence of N-ethylmaleimide or menadione. This effect follows a change of state in the cytoplasm. It may not be due to reaction with SH groups. When granule movement is prevented in this way the subsequent addition of Ca(2+) and ATP does not induce the secretion of the proteins of the granules. 2. Menadione or iodoacetate stimulates some effects of suboptimum amounts of leucocidin. This effect probably follows a reaction with SH groups. 3. Flavine mononucleotide inhibits some effects of suboptimum amounts of leucocidin. 4. Leucocidin decreases the stimulation of glucose oxidation due to menadione but increases that due to flavine mononucleotide. Leucocidin decreases the adsorption of menadione by leucocytes but increases that of flavine mononucleotide. 5. The redox state of the nicotinamide-adenine nucleotide coenzymes is not altered during leucocidin action and flavine mononucleotide and menadione do not undergo significant continuous oxidation and reduction when added to the leucocyte.     

Mechanism of action of the inhibition of pyridine-nucleotide-dependent flavine enzymes using the systemic fungicide Dexon]

The actions of Dexon on the NADH-ferricyanide oxidoreductase and the NADPH oxidase system of electron transfer particles (ETP) from beef heart as well as on the NADPH-cytochrome c oxidoreductase from brewer's yeast (Saccharomyces carlsbergensis Hansen) were investigated. The inhibition of the NADH dehydrogenase activity of ETP and that of the yeast enzyme correspond with respect to the following characteristics: 1) increase in the inhibition, 2) enhancement of the Dexon sensitivity by one order of magnitude after preincubation in the presence of NAD(P)H, 3) irreversibility of the inhibition, 4) no detectable changes in the spectral properties and in coenzyme activity of FMN after acid extraction from Dexon-treated enzyme. The inhibition of the NADH dehydrogenase activity of ETP is diminished by both NAD+ and FMN. However, no interaction of Dexon with NAD(P)H or FMN could be detected in the absence of enzyme or apoenzyme. The concentration of half-inhibition by Dexon for the yeast enzyme corresponds with its FMN concentration. It is proposed that both apoenzyme, NAD(P)H and FMN are involved in the interaction with Dexon. Possible mechanisms of binding are both complanar complexations of the ring systems and a triazene formation between FMNH2 and Dexon. The NADPH oxidase activity of the ETP is partly inhibited; the share inhibited by Dexon may represent the pathway via the transhydrogenase reaction.     

Rat kidney L-alpha-hydroxy acid oxidase: isolation of enzyme with one flavine coenzyme per two subunits.

L-alpha-Hydroxy acid oxidase (listed as EC 1.4.3.2, L-amino acid: O2 oxidoreductase) has been purified 100-fold from rat kidney to apparent homogeneity by gel electrophoresis. A subunit molecular weight of 47,500 was found by sodium dodecyl sulfate gel electrophoresis, but in contrast to previous reports, the enzyme has been found to have a molecular weight of ca. 200,000 by Sephadex gel filtration and by dodecyl sulfate gel electrophoresis of the enzyme cross-linked with dimethyl suberimidate. A somewhat higher value was found by sedimentation equilibrium, but a tetrameric structure for the active enzyme is definitely established. The enzyme was found to contain the FMN coenzyme at a concentration of one FMN/102,000 daltons or one flavine/two subunits, a highly unusual finding. This ratio was determined from spectroscopic analysis of the FMN in lyophilized samples of the enzyme and by titration of the coenzyme with the flavine specific enzyme inactivator 2-hydroxy-3-butynoate. The enzyme has the same specific activity as a crystalline sample of the enzyme reported to have twice as much flavine/milligram.