EPR spectrometry of cytochrome P4502B4: Effects of mutations and substratebinding

The EPR spectra of NH2-terminal-truncated P450 cytochrome 2B4 and of severa l active site mutants that were previously shown to be profoundly altered i n catalytic properties were determined. From these spectra it was seen that the truncated P450 2B4, like the full length cytochrome, exists as the low spin ferric form, but upon mutation of threonine 302 to alanine approximat ely 40% of the cytochrome is present as the high spin ferric form (g approx imate to 8, 4, 2). A similar situation was observed in the double mutant E3 10L T302A, but not in the single mutant E301L. A rhombic high spin signal ( g approximate to 8, 4, 2) was observed when a substrate such as styrene, be nzphetamine, or cyclohexane was added to the truncated cytochrome. Accompan ying this change was the appearance of a signal at g = 1.98. Conversely, an axial high spin signal was observed (g approximate to 6, 6, 2) when cycloh exanecarboxaldehyde or 3-phenylpropionaldehyde was added to the truncated P 450 2B4. (C) 2000 Academic Press.