Putidaredoxin (PdX), the physiological effector of cytochrome P450(cam) (P450(cam)), serves to gate electron transfer into oxy-P450(cam) during the catalytic cycle of the enzyme. Redox-linked structural changes in PdX are necessary for the effective P450(cam) turnover reaction. PdX is believed to be difficult to be replaced by an artificial electron donor in the reaction pathway of P450(cam). We demonstrate that the catalytic cycle of wild-type P450(cam) can be supported in the presence of an artificial reductant, potassium ferrocyanide. Upon rapid mixing of ferrocyanide ion with P450(cam) we observed an intermediate with spectral features characteristic of compound 1. The rate constant for the formation of compound I in the presence of ferrocyanide supported reaction cycle was found to be comparable to the ones observed for H2O2 supported compound I formation in wild-type P450(cam) but was much lower than those observed for classical peroxidases. The results presented in this paper form the first kinetic analysis of this intermediate for an artificial electron-driven P450(cam) catalytic pathway in solution. (c) 2005 Elsevier Inc. All rights reserved.