Low Temperature EPR Spectroscopic Characterization of the - Interaction of Cytochrome P - 450 , - with a Spin Label Analog of Metyrapone

The interaction between cytochrome P-450,and a spin-labeled analog (l-oxyl-2,2,6,6-tetramethyl-4-piperdinyl nicotinate; RNO) of the inhibitor metyrapone (2-methyl-1,2-di-3-pyridyl-l-propanone) has been studied by both optical absorbance and EPR spectroscopy. RNO was shown to bind to cytochrome P-450,,, competitively with the substrate camphor. In buffer with and without detergent, optical absorbance spectroscopy was used to measure the binding constant for camphor and the ratio of this value for RNO and camphor. The calculated value for RNO in the presence of detergent is 1.9 2 0.2 X IO4 M-’. In the presence of detergent, the concentration of RNO, measured by EPR, obeyed a simple binding equation at both 300 K and 103 K, and KRNO (1.2 k 0.2 and 1.8 rt; 0.3 X lo4 M-’) calculated from these data agreed well with the value determined optically. Measurement of the concentration of the RNO-cytochrome P-450,complex using EPR spectroscopy at 103 K confirmed the value of KWO. At 103 K, asymmetric doublets are present in the “cytochrome P-450” region of the EPR spectrum of the RNO-cytochrome P-450,,, complex and are consistent with a dipole-dipole interaction between the paramagnetic ferric ion and the nitroxide residue of the spin label. Computer simulation of this part of the EPR spectrum of the RNO-cytochrome P-450,,, complex precisely reproduced the experimental spectrum when the nitroxide residue was assumed to be 5.7 & 0.2 A from the hemin iron. These results are consistent with the RNO being bound to the hemin iron via the pyridine nitrogen and motion of the remainder of the molecule being relatively restricted.