NAD* radical

It has been suggested that the electrochemical oxidation of NADH may be further complicated by oxidation routes other than the one described by scheme (4) (42,45). Possible routes involving the intermediate radicals in scheme (4) are the oxidation of dimers formed from NAD radicals or the disproportionation reaction ... 

The formal potential of the NAD+/NADH redox couple is -0.56 V vs. SCE at pH 7 [15, 17]. However, at platinum and glassy carbon electrodes NADH, oxidation occurs at 0.7 V and 0.6 V vs. SCE, respectively [18]. From these oxidation potentials, it is clear that the direct electrochemical oxidation of NADH requires a substantial overpotential. In nature, NADH oxidation is thought to occur by a one-step hydride transfer. However, on bare electrodes the reaction has been shown to occur via a different and higher energy pathway which produces NAD radicals as intermediates. 

The oxidation of the dimer (reaction 13) has been thermodynamically and Id-netically studied with synthetic analogues of NAD dimers at gold and platinum electrodes [405] because (NAD)2 shows interest as a potential two-electron donor acting with no involvement of proton transfer, and its oxidation may provide a way of generating the NAD radical under milder conditions than that depicted in Sch. 1. Additionally, (NAD)2 may be able to participate in enzymatic reactions [406-408],... 

The NAD+ and (NAD)2 analogs form a chemically reversible redox couple but with a large separation (0.7 — 1.2 V) between the electrochemical reduction of NAD+ and the oxidation of (NAD)2. It has been suggested that the reason for this peak separation lies in the different rate-limiting step for both processes. In fact, the reduction of NAD+ to (NAD)2 requires the irreversible radical-radical dimerization of two NAD radicals, whereas the oxidation of (NAD)2 into 2NAD+ demands the cleavage of the dimerization radical (NAD)2 into NAD and NAD+ (Sch. 1). The reduction of NAD+ is Idnetically controlled by the dimerization of the NAD radicals, while the oxidation of (NAD)2 involves mixed kinetic control by electron transfer and cleavage of the (NAD)2 " " radical or total kinetic control by electron transfer [405]. 

Direct, nonmediated electrochemical reduction of NADIP)" " at modified electrode surfaces has been used to produce the en2ymatically active NAD(P)H and even to couple the NAD(P)H regeneration process with some biocatalytic reactions [228]. The modifier molecules used for these purposes are not redox active and they do not mediate the electron-transfer process between an electrode and NAD(P)+ however, they can effectively decrease the required overpotential and prevent formation of the nonenzymatically active dimer product [228]. For example, the efficiency of the direct electrochemical regeneration of NADH from NAD" " was enhanced by the use of a cholesterol-modified gold amalgam electrode that hinders the dimerization of the NAD-radicals on its modified-surface [228]. This direct electrochemical NAD+ reduction process was used favorably to drive an enzymatic reduction of pyruvate to D-lactate in the presence of lactate dehydrogenase. The turnover number for NAD" " was estimated as 1400 s k Other modifiers that enhance formation of the enzymatically active NAD(P)H include L-histidine [229] and benzimidazole [230], immobilized as monolayers on silver electrodes. CycKc voltammetric experiments demonstrated that these modified electrodes can catalyze the reduction of NAD+ to enzymatically active NADH at particularly low overpotentials. 

The calculated redox potential of the NAD- radical (X max 400 nm) in the excited stated , as follows ... 

Generation of H2O2 by this pathway would account for the lack of a difference in rates of photooxidation of the dimer in the presence or absence of oxygen. Generation of only traces of H2O2 by NADH irradiated under anaerobic conditions confirms the role of the NAD- radical in generation of H2O2 under such conditions, since photoionization of NADH leads to production of about 10% NAD . 

Because irradiation of the dimer (NADP)2 also leads to the appearance of NADPH ( 10% of the initial dimer concentration), this may lead to involvement in the photoreduction reaction of the NAD- radical (Scheme 2, below. Equation (lA, 1C) or disproportionation of the dimer in the excited state (Equation IB) or both. The formation of radicals during photodissociation of (NAD)2 at pH 9.5 by pulsed N2 laser excitation at 337 nm has been confirmed by Reverse Pulse Polarography . 

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