EPR STUDIES ON THE PHOTOPRODUCTS OF FERRIC CYTOCHROME P450CAM (CYP101) NITROSYL COMPLEXES - EFFECTS OF CAMPHOR AND ITS ANALOGS ON LIGAND-BOUND STRUCTURES

Photolyzed products of the NO complexes of ferric cytochrome P450cam b oth in the substrate-free and several substrate-bound states were trap ped and examined by EPR spectroscopy at 5 K. In the absence of substra te, the photoproduct exhibited ferric high- and low-spin signals, neit her of which showed the line-width broadening characteristic of magnet ic interaction between photodissociated NO and the heme iron. This fin ding indicates that photodissociated NO can diffuse to the unconstrain ed distal heme pocket giving the high-spin heme, and that a part of th e high-spin heme species converts to the low-spin heme upon coordinati ng an aqua molecule. When a substrate, camphor or adamantanone, was bo und at the site above the heme, the photoproduct exhibited widespread EPR absorptions together with a new distinct signal at g similar to 4. 4. The new signals are assignable to a weakly spin-coupled species bet ween 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 co mplex exhibited a spin-coupled EPR signal at g similar to 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 ad amantane-bound state only yielded low-spin signals, and exhibited no s pin-coupled signals. This result suggests that adamantane is mobile in the substrate pocket due to the lack of hydrogen bond formation with Tyr96. Thus, EPR examinations of the photolyzed NO complex at 5 K prov ide information on the constraints of the distal heme cavity. Based on the results, the structure of the heme vicinity in cytochrome P450cam is discussed.