Photo-cytochrome b5 - A New Tool to Study the Cytochrome P450 Electron-transport Chain

To study the enigmatic role of cytochrome b(5) in a cytochrome P450 monooxygenase multienzyme system, we expressed and purified a photoreactive analogue of cytochrome b(5), having methionine residues substituted with photolabile diazirine derivatives (photo-cyt b(5)). An equimolar mixture of cytochrome P450 (CYP) 2B4 and photo-cyt b(5) was photolyzed and products separated using SDS-PAGE. Several oligomers composed of CYP2B4 and cyt b(5) (MALDI-TOF/TOF mass spectrometry) in 1: 1, 1: 2, and 2: 1 molar ratios have been found and their covalent nature confirmed by MALDI-TOF/TOF mass spectrometry of chymotryptic peptides. The amount of oligomers formed was markedly increased by the presence of the CYP2B4 substrate, diamantane, showing that the substrate binding increases the affinity between cytochromes P450 and b(5).