Mechanism of O₂ Activation by Cytochrome P450cam Studied by Isotope Effects and Transient State Kinetics

Abstract

The early steps in dioxygen activation by the monooxygenase cytochrome P450cam (CYP101) include binding of O2 to ferrous P450cam to yield the ferric-superoxo form (oxyP450cam) followed by an irreversible, long-range electron transfer from putidaredoxin to reduce the oxyP450cam. The steady state kinetic parameter kcat/Km(O2) has been studied by a variety of probes that indicate a small D2O solvent isotope effect (1.21 +/- 0.08), a very small solvent viscosogen effect, and a 16O/18O isotope effect of 1.0147 +/- 0.0007. This latter value, which can be compared with the 16O/18O equilibrium isotope effect of 1.0048 +/- 0.0003 measured for oxyP450cam formation, is attributed to a primarily rate-limiting outer-sphere electron transfer from the heme iron center as O2 that has prebound to protein approaches the active site cofactor. The electron transfer from putidaredoxin to oxyP450cam was investigated by rapid mixing at 25 degrees C to complement previous lower-temperature measurements. A rate of 390 +/- 23 s-1 (and a near-unity solvent isotope effect) supports the view that the long-range electron transfer from reduced putidaredoxin to oxyP450cam is rapid relative to dissociation of O2 from the enzyme. P450cam represents the first enzymatic reaction of O2 in which both equilibrium and kinetic 16O/18O isotope effects have been measured.

Synergistic Effects of Mutations in Cytochrome P450cam Designed To Mimic CYP101D1

“…KSIE for the wild type P450cam was in agreement with previous studies of P450cam. 36,38,39 In our measurements (Table 2), none of the mutants showed any significant increase in the KSIE. The presence of 400 mM KCl did not alter the isotope effect measurements.…”

Identification of new substrates for the CYP106A1‐mediated 11‐oxidation and investigation of the reaction mechanism

“…The product structures confirmed the 11-oxidation of all substrates; however, only the 11-OH-AD conversion led to a single 11-keto-product, whereas prednisolone and dexamethasone displayed several hydroxy-metabolites as well. As a result, the regio-selective 11-oxidation of 11-OH-AD was chosen to study the reaction mechanism employing the KSIE, a proven method to distinguish the involvement of P450 reaction intermediates Cpd I or the ferric peroxoanion during P450 catalysis [1,18,21,31,38,47,49].…”

Measurement of oxygen isotope ratios (18O/16O) of aqueous O2 in small samples by gas chromatography/isotope ratio mass spectrometry

“…[6][7][8][9] On the molecular level, fractionation of stable O isotopes is used as a probe for identification of electron and proton transfer reaction steps as well as O-O bond cleavages in the enzymatic activation of molecular oxygen. [10][11][12] The 18 Okinetic isotope effects ( 18 O-KIEs) of O 2 in these reactions reveal the identity of reactive oxygen intermediates in various enzymes [13][14][15][16] and recent work suggests that knowledge of 18 O-KIEs could improve our understanding of oxygenation mechanisms of organic pollutants, which is an important detoxification process. [17,18] However, sensitive methods for a simple and robust measurement of O isotope ratios in small samples of dissolved O 2 by isotope ratio mass spectrometry (IRMS) are currently lacking.…”