Yann Astier

The electrochemistry and scanning tunnelling microscopy of the flavoprotein putidaredoxin reductase on alkanethiol-modified gold

AbstractCyclic voltammetry studies of putidaredoxin reductase (PdR) were carried out on 1-hexanethiol- and 1-dodecanethiol-modifiedgold electrodes, and the effect of NAD /NADH were investigated. Electron transfer rate constants were calculated for bothelectrode modifiers, and a catalytic reduction current was observed in the presence of NAD at dodecanethiol-modified goldsurfaces. Complementary in situ scanning tunnelling microscopy images of the electrode surface at high resolution were obtained influid which show that the PdR molecules are immobilised in a highly uniform manner on the modified gold surface. The resultssuggest that the immobilised enzyme is both electrochemically and catalytically active.# 2003 Elsevier Science B.V. All rights reserved. Keywords: Cyclic voltammetry; Scanning tunnelling microscopy; Putidaredoxin reductase; Self assembled monolayers 1. IntroductionIn 1977 the work of Eddowes and Hill [1,2], showedthat the problem of slow electron transfer between anelectrode and a metalloprotein is very often due touncontrolled immobilisation and this could be overcomeby the use of ‘promoters’ or ‘facilitators’ bound to theelectrode surfaces. These compounds bind at the elec-trode surface but also contain groups that are capable ofinteracting with amino acid side chains on the surface ofthe protein or enzyme. Since then much work has beenfocussed into developing suitably mediated or modifiedelectrode systems, which facilitate biological electro-chemistry [3,4]. This has been motivated by a funda-mental need to understand the mechanism of biologicalredox reactions. A major step towards understandingthe role of the promoters and the electron transferreactions of cytochrome c was reported in 1981 byAlbery et al. [5].Recently there have been studies of many selfassembled monolayers (SAMs) of thiols, investigatedby both electrochemistry [6] and scanning tunnellingmicroscopy (STM) [7]. These SAMs have been found tofacilitate direct electron transfer at modified surfaces.The compounds bind to gold, via sulphur-containingresidues, but also contain residues which are capable ofinteracting with groups on the surfaces of the proteinsand enzymes.Putidaredoxin reductase (PdR) is one of three pro-teins involved in the catalytic cycle that allows stereo-selective oxidation of camphor to 5-exo-hydroxycamphor. This reaction is catalysed by thehaem monooxygenase cytochrome P450cam. PdR con-tains one flavin