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We have used laser flash photolysis to investigate the kinetics of oxidation of reduced plastocyanins obtained from spinach and the green alga Monoraphidium braunii by the triplet states of lumiflavin, riboflavin and FMN. We have compared the results of these experiments with the kinetics of reduction of the oxidized forms of these proteins by the corresponding flavin semiquinones, as well as with the kinetics of flavin oxidation and reduction of cytochrome c552 (Class I c-type cytochrome, generic name c553) from Monoraphidium. in all cases, the rate constants for oxidation were one or two orders of magnitude larger than for reduction, consistent with the greater thermodynamic driving force for the oxidation reaction. Similar steric and electrostatic effects were observed for both reactions with all proteins, suggesting that the same (or closely adjacent) sites were being utilized for electron removal and entry. The two algal proteins were quite similar to one another in their redox properties, consistent with their physiological role of being able to substitute for one another in photosynthetic electron transport. In contrast, the algal plastocyanin was more reactive than the spinach protein in both oxidation and reduction, suggesting differences in their steric properties at the site of electron transfer. [ABSTRACT FROM AUTHOR]

Hexose oxidase from the red seaweed, Chondrus crispus was purified to homogeneity: The enzyme appeared to be encapsulated in particles obtained alter mechanical disintegration of the fronds. Liberation of the enzyme in soluble form required either waiting for the spontaneous development of a suitable microbial flora in the suspension, or treatment with a mixture of proteases (pronase). As deduced from (SDS/)PAGE, the enzyme has a molecular mass of 87 kDa and probably consists of subunits of 36 kDa and 25 kDa. The low isoelectric point of 2.8 and the presence of 25 % (by mass) sugars indicate that the enzyme is a strongly acidic glycoprotein. The absorption spectrum of isolated enzyme minus that of the substrate-reduced enzyme, and the EPR spectrum of the free radical observed in the reduced enzyme revealed the presence of a flavin. This cofactor is probably covalently bound since flavins were not released upon denaturation of the enzyme by heat or acid treatment. Taking free FAD as a reference compound, the enzyme contains 1 mol flavin/mol enzyme. EPR spectroscopy of the purified preparation showed the presence of Cu2+. However, since the amount was substoichiometric, substrate addition did not affect the signal, and the addition of chelator or Cu2+ did not affect the activity, the presence of this metal ion seems adventitious. It is concluded that the large discrepancies between the presently and the previously reported [Sullivan, J. D. & Ikawa, M. (1973) Biochim. Biophys. Acre 309, 11–22] characteristics of the enzyme probably originate from the characterization of a contaminating protein in the latter case. [ABSTRACT FROM AUTHOR]

The behaviour of cytochrome b5 reductase holoenzyme and apoenzyme toward blue-dextran-Sepharose has been studied. Holoenzyme was adsorbed at low ionic strength and could be eluted with 100 μM NADH or NAD+. Flavin-free enzyme was even more strongly bound and could be eluted with 1 M NaCl, or 100 μM NADH + 10 μM FAD. Separately the cofactors were without effect. FMN was less effective than FAD. ADP and AMP eluted nothing. Cibacron blue F3GA was found to exert a mixed inhibition on NADH oxidation. Dye binding to holoenzyme elicited a characteristic red shift in its spectrum. Comparison of the difference spectrum amplitudes at 680 and 585 nm showed the presence of a second binding mode at higher dye concentrations. These results point to the existence for cytochrome b5 reductase of two binding sites with high affinity for blue-dextran-Sepharose: the NADH binding site and the flavin binding site. For the latter it is clear that the isoalloxazine pocket must play a role in dye binding. Cytochrome b5 reductase is the second flavoenzyme which has been shown to have affinity for immobilized dye at the flavin site, the first one being flavocytochrome b2, an FMN-dependent enzyme [D. Pompon and F. Lederer (1978) Eur. J. Biochern. 90, 563–569]. [ABSTRACT FROM AUTHOR]

Evidence is given for the fact that the excited flavin triplet (3Flox*) exhibits competitive 1e- and 2e- transfer chemistry, depending on the nature of the photosubstrate. As an ‘external’ photoreductant, the 2e- donor borohydride has been investigated. Borohydride is found to compete effectively with the ‘internal’ 1e- donors, namely excess starting flavin in the ground state (Flox) and, as primary product, (alkyl)dihydroflavin (RFlredH). It will be shown that some of the flavin radicals, observed by earlier authors in the reaction of 3Flox* with CH or C-COO- substrates or in the autophotolytic side-chain cleavage of riboflavin, are due to the dye-dye reaction (I): [This symbol cannot be presented in ASCII format] In contrast flavin photoreduction by borohydride or hydrocarbon substrates need not involve radicals, but may in fact be a hydride or ‘carbanion-plus-proton’ addition towards the highly polar and considerably basic (pK = 4.4) acceptor triplet (cf. reaction II) [This symbol cannot be presented in ASCII format] The products are much more photoreactive than the starting substrates, which leads to the secondary photocomproportionation (III): [This symbol cannot be presented in ASCII format] This latter reaction is the second source of radicals in the system. This (cf. II) ‘photohydrogenation’ of flavin is mechanistically related to the biological reduction of flavin by CH substrates. [ABSTRACT FROM AUTHOR]

The use of DL-[2-2H]lactate in steady-state measurements of ferricyanide reduction by flavocytochrome b2 at 30°C has previously yielded an isotope effect of 5 [F. Lederer (1974) Eur. J. Biochem. 46, 393–399]. We report here studies carried out at 5°C with L-[2-2H]lactate, where fiavin and heme reduction were observed in the stopped-flow apparatus, in the absence of acceptor. The generally biphasic reduction curves were analysed according to a new mathematical treatment which allowed us to derive microscopic constants from initial reduction rates. It has thus been possible to determine an isotope effect of 8 on flavin reduction, 6 on heme reduction, compared to 4 in the steady state. Consequently, two slightly rate-limiting steps occur after the first one where the α-hydrogen is abstracted. It has also been possible to calculate the substrate association and dissociation rate constants for intact enzyme. The studies were carried out in parallel on intact and cleaved cytochrome b2. The results suggest that proteolysis affects essentially the steps involved in flavin reduction, and not intramolecular electron transfer steps. Moreover, the experimental data obtained at low rates of electron entry have led us to reexamine a previously proposed scheme for electron transfer [Capeillère-Blandin, Bray, Iwatsubo and Labeyrie (1975) Eur. J. Biochern. 54, 549–566]. An alternative model based on computer-simulation studies will be presented in a paper in this journal. [ABSTRACT FROM AUTHOR]

The catalytic action of 5-deazaflavin in the photochemical reduction of flavin and iron proteins [Massey, V. and Hemmerich, P. (1978) Biochemistry, 17, 9-17] is shown to be due to the highly reactive 5-deazaflavosemiquinone. This radical is generated in a complex sequence of reactions, which involves (a) covalent photoaddition of the substrate residue to the deazaflavin, (b) fast secondary photoreaction of this adduct with starting deazaflavin to yield a covalent radical dimer, accompanied by the liberation of the oxidized substrate, and (c) deazaflavin-sensitized cleavage of the radical dimer to the monomers. The structure and properties of this radical (redimerisation or dismutation) and the precursor intermediates as well as the mechanism of the photoreaction are described. Deazaflavins and their natural parent compounds are compared with respect to their different redox behavior and radical stability. The syntheses of 5-deuterated deazaflavins are described and their redox reactions are compared with those of normal deazaflavins. [ABSTRACT FROM AUTHOR]

By sulfur substitution of the N-5 atom in flavins and flavocoenzymes a flavin analog is obtained, 5-thiaflavin, which is found to be isoelectronic and isosteric with natural flavin in the fully reduced and half-reduced states, but not in the oxidized state. Among the three 'redox shuttles' characterizing the flavin system, viz. upper 1e-, lower 1e- and 2e- shuttle, only the second one is retained in thiaflavin, which limits the redox activity of this system to 1e- transfer. The structure and properties of the molecular species participating in the thiaflavin redox system are discussed in comparison with the flavin system. The corresponding chemistry of a '2e- flavin', 5-deazaflavin, has been treated in the preceding paper. 5-Thiaflavin is found to exhibit a stable neutral radical, which is analogous to the 'blue' flavosemiquinone. Unlike normal flavin, where the radical is in a dismutation equilibrium, thiaflavin radical shows reversible formation of a covalent dimer, which is stable in aprotic solution and disproportionates only in water, with irreversible formation of a sulfoxide. The ultraviolet and infrared spectra of the dimer are in agreement with the structure of two 5-thiaflavin molecules linked covalently at their 4a carbons. This corroborates the earlier hypothesis that the essential intermediate in the dismutation of normal flavin is likewise a covalent dimer. Thiaflavin is tightly bound by apoflavodoxin. The protein catalyses the autoxidation to the radical state. Thiaflavodoxin radical is even more stable (towards further oxidation) than is the free thiaflavin radical. The redox potential of the couple reduced thiaflavin/thiaflavin radical (sFlred/sFl) is surprisingly high. From the reversible equilibrium established with ferricyanide, sFlred + Fe(CN)63- ⇄ sFl• + Fe(CN)64-, the standard potential of the sFlred/sFl• couple, Em at pH 7, has been estimated as + 0.38 V. [ABSTRACT FROM AUTHOR]

Simulation studies by digital computer were undertaken in order to test and clarify the interpretations deduced from experimental data concerning the electron transfer mechanism from L-lactate to flavocytochrome b2, which were presented in a preceding paper in this journal. The reaction scheme proposed as the ‘best’ one is composed of 7 steps. It allows the best fitting of the time courses established for the oxidized ravin (Flox), the ravin semiquinone (Flsq), the fully reduced ravin (Flred), and the reduced haem (Hred); it can be extended to 1 s. This scheme also allows a good simulation of the general shape of preequilibrium titration curves obtained at a 200-ms reaction time for Hred and Flsq, and a valuable simulation of the reduction electron paramagnetic resonance time course established for Hred and Flsq at low lactate concentration. The agreement between experimental and simulated curves led to an estimation of some rate constants experimentally unknown, relative (in particular) to the electron exchange between ravin and haem and between couples of flavins. Another interest of these stimulation studies was to point out the obligatory involvement of a slow final step to perform the flavocytochrome b2 full reduction; this step could be controlled by some conformational change of the protein. [ABSTRACT FROM AUTHOR]