Vol. 70, No. 3, 1976 BIOCHEMICAL AND BIOPHYSICAL RESEARCH COMMUNICATIONS SPECTRAL INTERMEDIATES IN THE REACTION OF OXYGEN WITH PURIFIED LIVER MICROSOMAL CYTOCHROME P-450 F. Peter Guengerich,? David P. Ballou, and Minor J. Department of Biological Chemistry Medical School, The University of Michigan Ann Arbor, Michigan 48109 Received March 25,1976 Coon Summary: Stopped flow spectrophotometry has shown the occurrence of two dis- tinct spectral intermediates in the reaction of oxygen with the reduced form of highly purified cytochrome P-450 from liver microsomes. As indicated by difference spectra, Complex I (with maxima at 430 and 450 nm) is rapidly formed and then de- cays to form Complex II (with a broad maximum at 440 nm), which resembles the in- termediate seen in steady state experiments. In the reaction sequence, P-450%'- *Complex I+Complex II+P-450@., the last step is rate-limiting. The rate of that step is inadequate to account for the known turnover number of the enzyme in benzphetamine hydroxylation unless NADPH-cytochrome P-450 reductase or cytochrome b is added. The latter protein does not appear to function as an electron carrier in this process. In 1971 Estabrook et al. (1) found a new spectral intermediate in liver mi- -- crosomal suspensions under steady state conditions which they attributed to an oxygenated form of reduced cytochrome P-450. They also observed changes in the extent of NADPH-dependent cytochrome 9 reduction which indicated that this heme- protein may serve as an electron donor to the oxygenated form of cytochrome P-450. Gunsalus et al --* (2) and Ishimura et al. (3) also found such a spectral intermedi- -- ate in the reaction of oxygen with the reduced form of highly purified cytochrome P-450cam from Pseudomonas puti da. RHsen and Stier (4) provided further evidence from flash photolysis experiments for the occurrence of the oxyferro complex in liver microsomes and showed that 02 and CO are bound competitively to the cyto- chrome. The mixed function oxidase of liver microsomal membranes was resolved in this laboratory into three components 1 (P-~~OLM, NADPH-cytochrome P-450 reductase, and phosphatidylcholine) (5-8), and the purification and characterization of the reductase (9) and of multiple forms of P-450~~ (10-12) have been reported more recently. The present paper describes rapid reaction and steady state spectral studies which demonstrate the occurrence of two distinct intermediates in the red. reaction of P-450~~ with molecular oxygen. The cytochrome preparations used tPostdoctora1 Fellow, United States Public Health Service. Present address, Department of Biochemistry, Vanderbilt University, Nashville, Tennessee. 'The following abbreviations are used: P-~~OLM, liver microsomal cytochrome P-450; P-450%* and P-450Gd*, oxidized and reduced forms of the cytochrome; and dilauroyl-GPC, dilauroylglyceryl-3-phospharylcholine. Copyright 0 1976 by Academic Press, Inc. All rights of reproduction in any form reserved. 951