Electrocatalytic Hydroxylation of Sterols by Steroid C25 Dehydrogenase from Sterolibacterium denitrificans

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.