The electrochemically driven catalysis of the complex molybdoenzyme steroid C25 dehydrogenase (S25DH) from the β-Proteobacterium Sterolibacterium denitrificans is reported. S25DH catalyses the oxygen-independent regioselective hydroxylation of the tertiary C25 atom of sterols and also their derivatives. Cholest-4-en-3-one is a native substrate for S25DH, which produces 25-hydroxycholest-4-en-3-one as a product of catalytic turnover. Cholecalciferol (vitD3 ) is also a substrate. S25DH was immobilised on a modified gold working electrode with the co-adsorbent chitosan. The complexes ferricyanide ([Fe(CN)6 ]3- ) and ferrocenium methanol (FM+ ) are effective artificial electron acceptors from S25DH and act as mediators of electron transfer between the electrode and the enzyme. 2-Hydroxypropyl-β-cyclodextrin (HPCD) was employed as a sterol solubiliser, in addition to 2-methoxyethanol. The catalytic activity varied, depending upon the concentration of solubiliser in the reaction mixture. Parallel studies with [Fe(CN)6 ]3- as a chemical (as opposed to electrochemical) oxidant coupled to HPLC analysis show that S25DH is capable of oxidising both vitD3 and its less stable isomer, pre-vitD3 , and that the former substrate is stabilised by HPCD.
Keywords: electrochemistry; electron transfer; enzyme catalysis; molybdenum; steroids.
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