Biocatalytic reduction mechanism

Venkitasubramanian, P., Daniels, L. and Rosazza, J.P.N., Biocatalytic reduction of carboxylic acids mechanism and application. In Biocatalysis in the Pharmaceutical and Biotechnology Industries, Patel, R. (ed). CRC Press LLC Boca Raton, FL, 2006, pp. 425-440. 

The clear detection of both reversible active-site and biocatalytic waves represents a completeness that establishes the feasibility of applying direct elec-tochemistry to probe the mechanism of action of complex redox enzymes. To take this further, I shall take up the author s prerogative for mentioning studies currently underway in the laboratory and mention, briefly, another membrane-bound enzyme, fumarate reductase (FR), isolated from Escherichia coll Structurally, this is closely related to the more familiar succinate dehydrogenase (SDH) which constitutes the major part of Complex II of the mitochondrial respiratory chain. Of the four subunits which make up the membrane-bound system, two may be freed to give a soluble enzyme that is active in fumarate reduction by artificial electron donors [230]. The larger of these, MW approx. 70000, contains, like SDH, a covalently bound FAD. The smaller, MW approx. 30000, appears to contain three Fe-S clusters. These are termed centre 1 ([2Fe-2S]), centre 2 ([4Fe-4S]) and centre 3 ([3Fe-4S]). Their respective reduction potentials as determined by potentiometry are — 20 mV, — 320 mV, and — 70 mV [231], (Although the potential of the FAD has not been determined for FR, the two-electron value for beef heart SDH is — 79 mV at pH 7.0. The radical form is unstable since the two one-electron reductions occur at potentials of — 127 and — 31 mV respectively [232].)... 

The role of centre 2, for which the potentiometric reduction potential, at — 320 mV, is too low to be involved directly in electron transport between hydroquinone and fumarate, is not clear [164,234,235]. In these electrochemical experiments we have so far detected neither its electrode response nor any change in the steady-state biocatalytic current as the potential is lowered beyond — 320 mV. Cammack and co-workers have proposed [234, 235] that this centre might be active in one-electron reduction and reoxidation of the more reducing FAD radical species that must be generated, albeit transiently, during turnover. Their mechanism of reduction of oxidized FAD in SDH is termed the dual-pathway model since the two single electrons flowing consecutively out of FAD do so by different routes. The lower potential pathway uses centre 2. An equivalent mechanism, with the direction of electron flow reversed, is applicable for FR. 

Some OYEs have the novel biocatalytic activity of reducing aliphatic sec-nitro compounds to carbonyl compounds instead of the expected amines. This process is the biocatalytic equivalent to the Nef reaction, and it spares the use of strong acids (like H2SO4) and N2O production of the chemical alternative. The bioreduction mechanism presumably proceeds by reduction of the nitro group to the nitroso group, which subsequently tautomerizes to the more stable oxime that is further reduced to an imine derivative, which spontaneously hydrolyzes to the carbonyl compound and ammonia (Scheme 2.15). 

However, the most extensively investigated class of ERs is members of the OYE family of flavin oxidoreductases (EC 1.6.99.1). There is detailed information known about OYEs, such as their structure, reaction mechanism, substrate scope, kinetic properties, and biocatalytic approaches. Therefore, this chapter will focus on this latter class of enzymes. They have been intensively studied over the past decade in view of their applicability in preparative-scale biotransformations [1, 8-12]. These FMN-containing enzymes catalyze the asymmetric reduction of a,p-unsaturated... 

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