Flavins reduction

Akiyama, S. K, and Hamme.s, G. G., 1981. Elementary. steps in die reaction mechani.sm of die pyruvate dehydrogenase mnltienzyme complex from Escherichia coli Kinetics of flavin reduction. Biochemistry 20 1491-1497. 

There is some evidence that the iron-sulfur protein, FhuF, participates in the mobilization of iron from hydroxamate siderophores in E. coli (Muller et ah, 1998 Hantke, K. unpublished observations). However, a reductase activity of FhuF has not been demonstrated. Many siderophore-iron reductases have been shown to be active in vitro and some have been purified. The characterization of these reductases has revealed them to be flavin reductases which obtain the electrons for flavin reduction from NAD(P)H, and whose main functions are in areas other than reduction of ferric iron (e.g. flavin reductase Fre, sulfite reductase). To date, no specialized siderophore-iron reductases have been identified. It has been suggested that the reduced flavins from flavin oxidoreductases are the electron donors for ferric iron reduction (Fontecave et ah, 1994). Recently it has been shown, after a fruitless search for a reducing enzyme, that reduction of Co3+ in cobalamin is achieved by reduced flavin. Also in this case it was suggested that cobalamins and corrinoids are reduced in vivo by flavins which may be generated by the flavin... 

In the transfer of reducing equivalents from the pyridine nucleotide pool, flavoproteins carry out a central role of mediating the conversion of the obligatory 2-electron reductant to 1-electron receptors such as hemes and iron-sulfur redox centers. In such a role, the semiquinone form of the flavin serves as a pivotal intermediate. The reduction of flavins and flavoproteins by reduced pyridine nucleotides has been extensively studied since the initial work of Singer and Kearney which showed that flavin reduction can occur in a non-enzyme catalyzed manner. The reduction proceeds as a 2-electron process since the formation of a nicotinamide semiquinone (a necessary intermediate in a 1-electron process) has been... 

Recent investigations have shed light on peculiarities of the NOS action mechanism the role of the H4B cofactor and CaM, and cooperativity in kinetic and thermodynamic properties of different components of the nitric oxide synthesis system. Stop flow experiments with eNOS (Abu-Soud et al., 2000) showed that calmodulin binding caused an increase in NADH-dependent flavin reduction from 0.13 to 86 s 1 at 10 °C. Under such conditions, in the presence of Arg, heme is reduced very slowly (0.005 s 1). Heme complex formation requires a relatively high concentration ofNO (>50 nM) and inhibits the entire process NADH oxidation and citrulline synthesis decreases 3-fold and Km increases 3-fold. NOS reactions were monitored at subzero temperatures in the presence of 50% ethylene glycol as an anti-freeze solvent (Bee et al., 1998). 

Comparison of Ratks for Overall Turnover with Rates OF Enzyme Flavin Reduction by Various Pyridine Nucleotides (361, 366) ... 

Nucleotide Turnover number k for flavin reduction (sec ) H rate/D rate ... 

The midpoint redox potentials for flavin reduction in PDR are E ox/sq = nl74mV for the first couple and E sq/red = n274mV for the second couple (Gassner et al., 1995). The potential for the 2Fe-2S center is E p = nl74 mV. Thus, the thermodynamic driving force favors reduction of the 2Fe-2S center, especially by the first electron transfer. The rate of intramolecular electron transfer for the reduced flavin to the oxidized 2Fe-2S center has been estimated at >200sec based on simulation of stopped flow kinetic data (Correll et ah, 1992). 

The data thus reveal that ionizable groups are responsible for controlling the rate of flavin reduction. The intermediate and slow kinetic phases of the reductive half-reaction with diethylmethylamine were observed to be essentially independent of substrate concentration above pH 7 below... 

Whiehever mechanism operates, it is clear that the rate of reduetion of the flavin group is totally limited by the cleavage of the aC-H bond sinee the deuterium kinetic isotope effect for this step is around 8 (Miles et al., 1992 Pompon et al., 1980). However, in flavocytochrome 2 the rate of flavin reduetion is some 6-fold faster than the overall steady-state turnover rate (Daff et al., 1996a). As a consequence the flavin reduction step eontributes little to the rate limitation of the overall catalytic cycle (Figure 3). In faet it is eleetron transfer from flavin-semiquinone to b2 -heme that is the major rate-determining step and this is discussed in the following seetion. 

Walsh et al. (52), as well as ourselves (53), argued that if a-proton removal were an obligatory step in flavin reduction, then substitution of a good leaving group X, such as chloride, at the )8-carbon of an amino acid oxidase substrate might reveal non-oxidative a-fi elimination of HX... 

The structures of EX and El were deduced by resolution of El into apoenzyme and free flavin-substrate adduct. The structure of this adduct was determined as 5-cyanoethy 1-1,5-dihydro FAD and that of EX was deduced to be a cationic imine resulting from elimination of NO2" from the initial 5-nitroethy 1-1,5-dihydro FAD adduct formed in the process controlled by k2 by nucleophilic attack of nitroethane carbanion on the position of oxidized flavin. The chemistry of flavin reduction by nitroethane carbanion at the active site of D-amino acid oxidase is given by the following scheme (Equation 19) in which the kinetically important... 

Binding of 4-hydroxybenzoate (S) in the phenolate form facilitates flavin reduction by NADPH. After NADP+ release, the flavin hydroquinone reacts with molecular oxygen to yield the flavin C4a-hydroperoxide oxygenation species. Protonation of the distal oxygen of the peroxflavin facilitates the electrophilic attack on the nucleophilic carbon center of the substrate phenolate. After monooxygenation, the resulting hydroxyflavin is... 

For PHBH to function as an efficient catalyst, the series of four conformational changes in a catalytic cycle have to be fast and coordinated compared with the chemical reactions of catalysis. For example, the observation that the reduction of flavin under optimal conditions for catalysis exhibits a full primary deuterium isotope effect (13) implies that the rate of reduction of flavin is limited by hydride transfer and not by conformational rearrangements. However, when the enzyme is stabilized in the in conformation (as with the mutant form, Ala45Gly), then a large fraction of flavin reduction becomes much slower under the same conditions and shows only a small deuterium isotope effect (21). 

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