The electron transfer reactivity of cytochrome P-450-CAM. Molecular oxygen dependent multiple turnovers using chemical reductants

Abstract

Cytochrome P-450, unlike most other cytochrome,s does not function merely as an electron carrier but is also an enzyme capable of catalyzing oxygenation reactions. This heme-containing monooxygenase activates molecular oxygen for insertion of one oxygen atom into organic substrates with concomitant reduction of the other oxygen atom to water. Bacterial P-450, isolated from camphor-grown Pseudomonas putida (P-450-CAM)†, utilizes molecular oxygen and NADH to hydroxylate camphor at the exo-5 position and initiate camphor degradation [1]. Because the hemoprotein itself cannot react directly with NADH, electrons are transferred from NADH to P-450-CAM via first a FAD-containing flavoprotein (putidaredoxin reductase, fp) and then an iron-sulfur protein (putidaredoxin. Pd). t001 Product Formation. a A. As a function of P-450-CAM Concentration P-450-CAM Conc. (μ M) Exo-5-hydroxycamphor formed c (nmol) With Putidaredoxin (3 μ M) 0 0.1 ± 0.01(2) 0.5 23.6 ± 1.0(2) 1.0 49.8 ± 1.5(6) 2.0 97.0 ± 2.5(2) Without Putidaredoxin 0 0.02 ± 0.01(2) 0.5 1.3 ± 0.1(2) 1.0 2.7 ± 0.2(3) 2.0 5.1 ± 0.2(3) B. As a Function of Time Incubation (min) Exo-5-hydroxycamphor formed c (Mol/Mol of P-450-CAM) With Putidaredoxin (3 μ M) 1 28.8(1) 2 49.8 ± 1.5(6) 5 101.7(1) 10 188.7 ± 5.0(2) Without Putidaredoxin 1 1.5 ± 0.1(2) 2 2.7 ± 0.2(3) 5 5.8 ± 0.1(2) 10 9.8 ± 0.3(2) C. Control Experiments Exo-5-hydroxycamphor formed c (nmol) Without Pd d With 5 μ M Pd e 1. No P-450 0.02 ± 0.01(2) 0.01 ± 0.01(2) 2. No NADH 0.01 ± 0.01(2) 0.01 ± 0.01(2) No PMS 0.02 ± 0.01(3) 0.01 ± 0.01(3) 4. Boiled P-450 f 0.03 ± 0.02(2) - 5. Myoglobin (no P-450) g None - bTwo minute incubations. a Incubations were done using optimized conditions unless otherwise indicated. Optimized conditions; 1 μ M P-450-CAM, 600 μ M d-camphor, 3 m M NADH, and 50 μ M PMS, in 20 m M phosphate buffer (pH 7.40, and 100 m M KCI), 2 ml total volume, with gentle oxygen bubbling. In the presence of Pd (3–5 μ M), 5 m M NADH was used. c The number in parentheses is the number of trials of a particular experiment. d Ten minute incubations without oxygen bubbling using optimized conditions except as follows: 1.6 m M NADH, 250 μ M PMS. e Same conditions as described in footnote d, plus oxygen bubbling. f P-450-CAM was boiled for 10 minutes prior to use. g 1 μ M myoglobin. of the inhibition and oxygen bubbling studies support the hypothesis that hydroxylation by the NADH/PMS/P-450-CAM system occurs by an oxygen-dependent enzymatic pathway. The role of the PMS in the system appears to be as a mediator of electrons from NADH to P-450. This system provides the first protocol for achieving molecular oxygen dependent multiple turnovers of P-450 in the absence of the fully reconstituted three protein system.