Concerning 1e- transfer in reduction by dihydronicotinamide: reaction of oxidized flavin and flavin radical with N-benzyl-1,5-dihydronicotinamide.

Abstract

The rates of reaction of 5-ethyl-3-methyllumiflavinium perchlorate and.5-ethyl-3-methyllumiflavinyl radical with a NADH analogue, N-benzyl-1,4-dihydronicotinamide, were measured anaerobically in tert-butanol and .5% acetonitrile/ 95% tert-butanol-solutions at 30C. The biphasic kinetics observed for the reaction of flavin radical with dihydronicotinamide were interpreted in terms.of both a leC and a 2e-mechanism; the former was found to be inadequate based on experimental requirements of the mechanism. The dihydronicotinamide reacts preferentially with oxidized flavin rather than flavin radicals even when the concentration of oxidized flavin. is at a concentration 5 orders of magnitude less than that of radical. These studies show that the "hydride" reduction of oxidized flavin by BNAH is more facile than is the le-reduction of flavin radical by BNAH.The oxidation of reduced pyridine nucleotides and their analogues is far from completely understood, especially when a flavin moiety is used as the oxidant as it is in the flavin coenzymes ofthe respiratory chain (1). Suelter and Metzler (2) studied the nonenzymatic reduction of several flavin analogues by 1-propyl-1,4-dihydronicotinamide and concluded that hydride transfer was occurring. Their deduction was based on the observations that: (i) electron-withdrawing.substituents on the. nicotinamide decreased the oxidation rate; (ii) the 4,4-dideutero-1,4-dihydronicotinamide reacted slower than its protio analogue by a factor of 0.31; and (iii) the oxidation rate increased with increasing solvent polarity. Radda and Calvin (3) pointed out that radical detection by ESR is not a useful. observation because the comproportionation reaction between oxidized flavinmononucleotide and reduced flavinmononucleotide (FMN and FMNH2, respectively) provides the flavin radical, FMNHF-.[1]Thus, the presence ofa flavin radical does not provide evidence for a le-transfer mechanism. In further studies (4-6), these results were confirmed and the N5 of the flavin. moiety was proposed to be the position accepting the hydride ion. The reaction mechanism for net hydride transfer. from dihydronicotinamides to flavin was later formulated to involve preequilibrium complex formation along the reaction path. This was based on the lack of correlation of log(kr te) for the redox reaction with the E112 potentials for a series of flavins and the good correlation between log(krale) with log(Ke) for complex formation ofthe flavins with tryptophan (7,8 (19)(20)(21)(22)(23)(24). A multistep rather than a single-step Htransfer has been suggested in order. to account for. the observations that product isotope effects have been found to be significantly larger than the kinetic ones. This observation-can be explained if an intermediate is formed along the reaction path for "hydride. transfer. " In most cases a pyridinyl radical was invoked. The direct determination of isotope ratios of products is inherently more difficult in the dihydronicotinamide reduction of flavins because of the exchangable nat...

Identification of the Melatonin-binding SiteMT 3 as the Quinone Reductase 2

“…Indeed, when hamster kidney membranes were prepared in the absence of detergent, MT 3 loosely associated with membrane components and fractionated almost equally in the pellet and in the supernatant of an ultracentrifugation. 3 This is consistent with the elution from the DEAE column at low ionic strength (30 -40 mM NaCl), and with the previously described hydrophobic properties of QR 2 (9,14,20,25). Third, the present QR 2 transfected (1-4) CHO cells.…”

“…This hamster/ human difference was also observed with the enzymatic data, where IC 50 were 1.5-20-fold higher with human QR 2 compared with hamster MT 3 /QR 2 . Besides, hamster MT 3 /QR 2 and human QR 2 had a similar Michaelis affinity for menadione and BNAH, with a K m of about 60 M for BNAH, comparable with the K m of NAD(P)H for QR 1 (20,23). Dicoumarol was confirmed as a poor inhibitor of QR 2 , with an IC 50 of about 600 M. Interestingly, the MT 3 compounds proved to be relatively good inhibitors of the enzyme, especially of the hamster preparation where the IC 50 of iodomelatonin and S26553 were in the micromolar range, well below the K m of the substrates.…”

“…Dicoumarol, a potent QR 1 inhibitor and poor QR 2 inhibitor (14) was added to the assay to ensure QR 2 specificity. Control assays of QR 1 activity were performed when necessary, using menadione as the substrate and NADH as the co-substrate, which provided good QR 1 specificity since NADH is a very poor co-substrate for QR 2 (14,20). 3 , Although, in the light of our results presented thereafter, MT 3 and QR 2 seem to designate a unique protein, for convenience in the present report, we alternatively refer to hamster MT 3 or MT 3 /QR 2 and human QR 2 , depending on the methodological approach involved.…”

Asymmetric azidohydroxylation of styrene derivatives mediated by a biomimetic styrene monooxygenase enzymatic cascade

“…Flavin reduction by NCBs such as BNAH is known to occur through direct hydride transfer in solution. 48,49 Different substituents were previously shown to affect the overall NCB redox potential and lead to different FAD reduction rates. 31 Therefore, we screened six NCBs bearing substituents varying from amide, acetyl, carboxylic acid to nitrile, with either a benzyl, butyl or alkyl amide chain on the nitrogen, and the classical Hantzsch ester (HEH, Figure 3E and 4), as a hydride donor for the reduction of FAD without interfering with the epoxidation.…”

Artificial Flavin Receptors: Effects of Hydrogen Bonding on Redox Properties of a Flavin Mimic

“…As the oxidation mechanisms for the oxidation of an NADH model and thiols by flavin are well established, they are pertinent for the kinetic study. The former proceeds via a hydride transfer to the N(5) position of an isoalloxazine ring (41,42,48), and the latter proceeds via nucleophilic attack of thiolate at the C(4a) position to form an adduct, followed by attack of the second thiolate at the sulfur atom of the adduct to give the corresponding disulfide and reduced flavin (25,26) as shown in Scheme 2.…”