Open AccessRedox properties of cytochrome P450 BM3 measured by direct methods
Author(s) -
Fleming Barry D.,
Tian Yanni,
Bell Stephen G.,
Wong LuetLok,
Urlacher Vlada,
Hill H. Allen O.
Publication year - 2003
Publication title -
european journal of biochemistry
Language(s) - English
Resource type - Journals
eISSN - 1432-1033
pISSN - 0014-2956
DOI - 10.1046/j.1432-1033.2003.03799.x
Subject(s) - chemistry , redox , heme , electron transfer , cytochrome , reductase , electron transport chain , hemeprotein , photochemistry , saturated calomel electrode , stereochemistry , inorganic chemistry , electrode , reference electrode , electrochemistry , biochemistry , enzyme
Cytochrome P450 BM3 is a self‐sufficient fatty acid monooxygenase consisting of a diflavin (FAD/FMN) reductase domain and a heme domain fused together in a single polypeptide chain. The multidomain structure makes it an ideal model system for studying the mechanism of electron transfer and for understanding P450 systems in general. Here we report the redox properties of the cytochrome P450 BM3 wild‐type holoenzyme, and its isolated FAD reductase and P450 heme domains, when immobilized in a didodecyldimethylammonium bromide film cast on an edge‐plane graphite electrode. The holoenzyme showed cyclic voltammetric peaks originating from both the flavin reductase domain and the Fe III /Fe II redox couple contained in the heme domain, with formal potentials of −0.388 and −0.250 V with respect to a saturated calomel electrode, respectively. When measured in buffer solutions containing the holoenzyme or FAD‐reductase domain, the reductase response could be maintained for several hours as a result of protein reorganization and refreshing at the didodecyldimethylammonium modified surface. When measured in buffer solution alone, the cyclic voltammetric peaks from the reductase domain rapidly diminished in favour of the heme response. Electron transfer from the electrode to the heme was measured directly and at a similarly fast rate ( k s ′ = 221 s −1 ) to natural biological rates. The redox potential of the Fe III /Fe II couple increased when carbon monoxide was bound to the reduced heme, but when in the presence of substrate(s) no shift in potential was observed. The reduced heme rapidly catalysed the reduction of oxygen to hydrogen peroxide.