Fluorescence properties of reduced flavins and flavoproteins

Fluorescence lifetimes and polarized emission properties of reduced flavin were measured using several model compounds and flavoproteins. Depending on the conditions of solvent and temperature or reduction method the lifetimes vary between 1 and 15 ns. The longer lifetime values are found in several forms of reduced lactate oxidase, in which a good correlation exists between fluorescence intensity and lifetime. In practically all flavoproteins the fluorescence is heterogeneous. Several mechanisms are proposed to explain the observed heterogeneity in lifetimes. The reduced models in glycerol at subzero temperature exhibit high degrees of polarization of the fluorescence, whereas distinct depolarization is encountered in several reduced flavoproteins suggesting a certain mobility of the flavin chromophor.     

The release of iron from horse spleen ferritin by reduced flavins

Ferritin-Fe(III) was rapidly and quantitatively reduced and liberated as Fe(II) by FMNH₂, FADH₂ and reduced riboflavin. Dithionite also released Fe(II) from ferritin but at less than 1% of the rate with FMNH₂. Cysteine, glutathione and ascorbate gave a similar slower rate and yielded less than 20% of the total iron from ferritin within a few hours. The reduction of ferritin-Fe(III) by the three riboflavin compounds gave complex second-order kinetics with overlapping fast and slow reactions. The fast reaction appeared to be non-specific and may be due to a reduction of Fe(III) of a lower degree of polymerization, equilibrated with ferritin iron. The amount of this Fe³⁺ ion initially reduced was small, less than 0.3% of the total iron. Addition of FMN to the ferritin–dithionite system enhanced the reduction; this is due to the reduction of FMN by dithionite to form FMNH₂ which then reduces ferritin-Fe(III). A comparison of the thermodynamic parameters of FMNH₂–ferritin and dithionite–ferritin complex formation showed that FMNH₂ required a lower activation energy and a negative entropy change, whereas dithionite required 50% more activation energy and showed a positive entropy change in ferritin reduction. The effectiveness of FMNH₂ in ferritin–Fe(III) reduction may be due to a specific binding of the riboflavin moiety to the protein portion of the ferritin molecule.     

Oxidation of pyruvic acid by flavins in the presence of amines

Pyruvic acid or other enolizable α-keto acids in the presence of primary or cyclic secondary amines in aprotic polar solvents efficiently reduce flavins and isoalloxazines to their 1,5-dihydro derivatives. The other product of this reaction is the diamide of citraconic acid (1). In the absence of flavin, the diamide of methylsuccinic acid (2) is obtained as the major product. The specificity for α-keto acid and amine, stoichiometric requirements, rates and quantities of CO₂ evolution, and the kinetics of the reaction were studied. On the basis of these data a mechanism is proposed that involves an enamine condensation followed by decarboxylation and then either reaction with flavin to give ultimately (1) and dihydroflavin, or reaction with an additional amine to give (2). The possible import of this kind of mechanism in biochemical systems is briefly discussed.     

Reaction of reduced flavins and flavoproteins with diphenyliodonium chloride

The reaction of diphenyliodonium chloride with free reduced flavins has been studied by stopped flow spectrophotometry under anaerobic conditions, and second order rate constants were determined as a function of pH. The reactive flavin species was identified as the reduced anion, based on an observed reaction pK of 6.7. The product mixture was independent of the initial concentration of reactant and contained approximately 20% oxidized flavin. The results can be modeled quantitatively on a modification of the mechanism proposed by Tew (Tew, D. G. (1993) Biochemistry 32, 10209-10215). The composition of the complex reaction mixture has been analyzed, and four flavin-phenyl adducts with distinctive absorbance and fluorescence characteristics have been identified, involving substitution at the flavin C4a, N5, and C8 positions. Inactivation of flavoprotein enzymes by diphenyliodonium has also been studied, and several examples were found where inactivation occurs readily, despite noninvolvement of radical intermediates in their reaction mechanisms. It can be concluded that inactivation by phenyliodonium species is not a valid indicator of catalytic mechanism involving radical intermediates. One of the several factors determining inactivation is maintenance of the enzyme flavin in the reduced form in the steady state of catalysis, the other factors being redox potential and accessibility of the inhibitor to the flavin active site.     

Uncoupling of the substrate monooxygenation and reduced pyridine nucleotide oxidation activities of salicylate hydroxylase by flavins

The NAD(P)H oxidation and substrate monooxygenation activities of Pseudomonas cepacia salicylate hydroxylase can be uncoupled by added flavins. The uncoupling is postulated to result from a reducing equivalent exchange between the hydroxylase-bound FADH₂ and the added flavins, leading to the reduction of the latter species and the regeneration of oxidized holoenzyme without hydroxylating the salicylate substrate. When exogenous FMN was added, the salicylate hydroxylase-catalyzed NAD(P)H oxidation could be coupled to the bacterial bioluminescence reaction, which is specific for fully reduced FMN as a substrate. The quantum yield of the coupled bioluminescence, based on the amount of NADH oxidized independently of salicylate monooxygenation, was determined to be 0.14 correlating closely with the known quantum yield of about 0.17 for reduced FMN in the luciferase-catalyzed bioluminescence reaction. A series of flavin derivatives were tested for their effects on the uncoupling of NAD(P)H oxidation and substrate monooxygenation activities of salicylate hydroxylase. Results indicated that the efficiency for interactions between the bound FADH₂ and free flavins was sensitive to the position of structural modification, size, and charge of the added flavin species, suggesting that the bound FADH₂ was partially exposed to aqueous medium under conditions of actual catalysis.     

Oxidation of cytochrome b5 reduced by ascorbic acid

The steady-state levels of aerobic and anaerobic reduction of cytochrome b5 by ascorbic acid and the initial rates of cytochrome b5 reduction in the presence of ascorbic acid and of anaerobic cytochrome P-450 reduction in the presence of NADH were used to calculate the rate constants for cytochrome b5 oxidation. The rate constant for cytochrome b5 autooxidation in the membrane is equal to that for isolated cytochrome b5, i. e., 5 X 10(-3) s-1 (37 degrees C). The rate constant for the second cytochrome b5 oxidation reaction in the membrane, i. e., electron transfer to cytochrome P-450, is equal to 140 X 10(-3) s-1 (37 degrees C).     

Flavokinase and FAD synthetase from Bacillus subtilis specific for reduced flavins.

A flavokinase preparation from Bacillus subtilis is described which catalyzes the phosphorylation of reduced, but not oxidized, riboflavin. The enzyme is distinguished from other known flavokinases also in having an unusually low Km for the flavin substrate, 50 to 100 nM. ATP is the obligatory phosphate donor; one ATP is utilized for each FMNH2 formed. Mg2+ or Zn2+ is required for the reaction; Co2+ and Mn2+ will substitute, but less effectively. The same enzyme preparation catalyzes the synthesis of FADH2 from FMNH2 and ATP, but not the synthesis of FAD from FMN and ATP. FADH2 is also formed from reduced riboflavin, presumably by sequential flavokinase and FAD synthetase action. Zn2+ cannot replace Mg2+ in FADH2 formation. The reverse reaction, formation of FMN from FAD, occurs only with reduced FAD, giving rise to FMNH2, and is dependent on the presence of inorganic pyrophosphate. The enzyme thus appears to be an FADH2 pyrophosphorylase. The two enzymatic activities, flavokinase and FADH2 pyrophosphorylase, although not separated during the purification procedure, are distinguished by differences in metal ion specificity, in concentration dependence for ATP (apparent Km for ATP = 300 microM for FADH2 synthesis and 6.5 microM for flavokinase), and in the inhibitory effects of riboflavin analogues.     

Flash photolysis of flavins. V. Oxidation and Disproportionation of flavin radicals

Flash photolysis techniques have been utilized to investigate the reactivity patterns of flavin radical species. Rate constants for disproportionation were found to be la the following order: lumiflavin>FMN>FAD and neutral radicals>anionic radicals. Neutral flavin radicals react with oxygen at a rate which is at least 10⁴ times slower than the anionic species. No evidence for an intermediate complex or adduct is obtained in this reaction. The pK values for the ionization of the neutral flavin radicals are in the order FAD>FMN>riboflavin=limiflavin. The rates of reaction of ferricyanide with flavin radicals are essentially independent of pH, whereas benzoquinone reacts slightly more slowly (5 times) with the neutral flavin radical than with the anionic form. Cytochromec reacts at least ten times more slowly with flavin radicals than does ferricyanide.     

Oxidation of reduced glutathione by subcellular fractions of rat liver

1. A new method was used to diminish the autoxidation of GSH. 2. The oxidation of GSH by liver homogenates was studied with regard to concentration of homogenate, concentration of GSH, time, pH and anaerobiosis. 3. GSH was oxidized by recombinations of the supernatant with microsomes and with mitochondria. Each fraction alone caused little oxidation. 4. Proteins in the supernatant were required to obtain the effect, and low-molecular-weight compounds in the same fraction increased its effect. 5. GSH diminished the formation of malonaldehyde in homogenates. 6. GSH prevented a stimulating effect of the supernatant on the formation of malonaldehyde in microsomes and in mitochondria. 7. The malonaldehyde formation in microsomes together with the supernatant did not start until the concentration of endogenous low-molecular-weight thiols had decreased to a low level. 8. It is suggested that part of the oxidation of GSH in homogenates is coupled to a mechanism that counteracts the peroxidation of membrane lipids.