Optical and EPR spectra have proved invaluable techniques in the investigation of P-450 cytochromes. A Type I difference spectrum (A,,,.= 385, A,,,,= 420 nm) characterizes the change from oxidized free enzyme to the enzyme substrate complex, while other ligands can produce the reverse transition (Type I1 difference spectrum, Am," 385, Am.= 420 nm). EPR spectra have shown that the former corresponds to a transition between a low-spin and a high-spin state of the heme and that the latter corresponds to a high-to low-spin transition: A third type of spectral change is produced when certain amines convert both high-and low-spin forms of cytochrome P-450 to an amine complex that exhibits a characteristic low-spin EPR spectrum.'The amine difference spectrum depends upon the initial proportion of high-and low-spin P-450 hemes and has been used to determine the spin state of cytochrome P-450.'

The activation of cholesterol side-chain cleavage by the action of ACTH is a key step in the stimulation of corticosteroid formation in the adrenal cortex? This paper will describe efforts to determine the effect of ACTH upon the cytochrome P-450 in adrenal mitochondria that is involved in this conversion.* Preseni address: Department of Biochemistry, University of Wisconsin, Madison,