Cytochrome P-450cam and putidaredoxin interaction during electron transfer.
Cytochrome P-450cam, the bacterial hemeprotein which catalyzes the 5-exo-hydroxylation of d-camphor, requires two electrons to activate molecular oxygen for this monooxygenase reaction. These two electrons are transferred to cytochrome P-450cam in two one-electron steps by the physiological reductant, putidaredoxin. The present study of the kinetics of reduction of cytochrome P-450cam by reduced putidaredoxin has shown that the reaction obeys first order kinetics with a rate constant of 33 s-1 at 25 degrees C with respect to: 1) the appearance of the carbon monoxide complex of Fe(II) cytochrome P-450cam; 2) the disappearance of the 645 nm absorbance band of high-spin Fe(III) cytochrome P-450cam; and 3) the disappearance of the g = 1.94 EPR signal of reduced putidaredoxin. This data was interpreted as indicative of the rapid formation of a bimolecular complex between reduced putidaredoxin Fe(III) cytochrome P-450cam. The existence of the complex was first shown indirectly by kinetic analysis and secondly directly by electron paramagnetic resonance spectroscopic analysis of samples which were freeze-quenched approximately 16 ms after mixing. The direct evidence for complex formation was the loss of the EPR signal of Fe(III) cytochrome P-450cam upon formation of the complex while the EPR signal of reduced putidaredoxin decays with the same kinetics as the appearance of Fe(II) cytochrome P-450. The mechanism of the loss of the EPR signal of cytochrome P-450 upon formation of the complex is not apparent at this time but may involve a conformational change of cytochrome P-450cam following complex formation.
D000693:Anaerobiosis; D002164:Camphor; D002248:Carbon Monoxide; D003577:Cytochrome P-450 Enzyme System / Q000378:metabolism; D004578:Electron Spin Resonance Spectroscopy; D004579:Electron Transport; D005288:Ferredoxins; D007700:Kinetics; D010084:Oxidation-Reduction; D011549:Pseudomonas / Q000378:metabolism
D005288:Ferredoxins; D002164:Camphor; D002248:Carbon Monoxide; D003577:Cytochrome P-450 Enzyme System