EPR Studies on the Photoproducts of Ferric Cytochrome P450cam (CYP101) Nitrosyl Complexes: Effects of Camphor and Its Analogues on Ligand-Bound Structures1

Abstract

Photolyzed products of the NO complexes of ferric cytochrome P450cam both in the substrate-free and several substrate-bound states were trapped and examined by EPR spectroscopy at 5 K. In the absence of substrate, the photoproduct exhibited ferric high- and low-spin signals, neither of which showed the line-width broadening characteristic of magnetic interaction between photodissociated NO and the heme iron. This finding indicates that photodissociated NO can diffuse to the unconstrained distal heme pocket giving the high-spin heme, and that a part of the high-spin heme species converts to the low-spin heme upon coordinating an aqua molecule. When a substrate, camphor or adamantanone, was bound at the site above the heme, the photoproduct exhibited widespread EPR absorptions together with a new distinct signal at g approximately 4.4. The new signals are assignable to a weakly spin-coupled species between the ferric heme iron and the photodissociated NO, indicating that the NO molecule is in close proximity to the heme iron by the steric crowding of the bound substrate. The photoproduct of the norcamphor complex exhibited a spin-coupled EPR signal at g approximately 5, in which the coupling is suggested to be weaker than that of the camphor-bound enzyme. On the other hand, the photoproduct of the NO complex in an adamantane-bound state only yielded low-spin signals, and exhibited no spin-coupled signals. This result suggests that adamantane is mobile in the substrate pocket due to the lack of hydrogen bond formation with Tyr96.(ABSTRACT TRUNCATED AT 250 WORDS)