Formation rates of the two ethyl isocyanide compounds of liver cytochrome P-450 at low temperatures.

Liver cytochrome P-450 forms two spectroscopically and functionally distinct compounds with ethyl isocyanide, but the structural basis for the difference between them is unknown. We have examined the relative rates of formation of the two compounds at low temperatures. Reduced cytochrome P-450 was generated photochemically from its carbon monoxide complex at temperatures below O’, and allowed to react with ethyl isocyanide. (a) The extent of conversion from the CO compound to the ethyl isocyanide compounds was a function of the relative concentrations of CO and ethyl isocyanide. At a concentration ratio of about 1, nearly complete conversion took place. (b) The ratio of the two ethyl isocyanide compounds formed upon irradiation with monochromatic light at 450 nm, measured by the relative heights of their Soret bands (455 and 430 nm), depended upon irradiation conditions such as temperature and time. In general, the lower the irradiation temperature, the higher the ratio of the 455-nm state to the 430-nm state. The sum of ethyl isocyanide compounds formed upon I-min irradiation, however, decreased with decreasing temperature below -30’; no conversion occurred at or below -85’. (c) On irradiation at -30°, the 455-nm state was produced rapidly at first, then its concentration decreased gradually while that of the 430-nm state increased. Thus, the ratio of the 455-nm state to the 430-nm state decreased with increasing irradiation time. (d) A slow reversion from ethyl isocyanide compounds to CO complex occurred as a dark reaction after irradiation. During this conversion, at -3O”, a rapid decrease in the ratio of the 455-nm state to the 430-nm state took place. These observations and others suggest that the following relationship exists among the cytochrome P-450 compounds studied.