Putidaredoxin-cytochrome P450(cam) interaction - Spin state of the heme iron modulates putidaredoxin structure

During the monooxygenase reaction catalyzed by cytochrome P450(cam) (P450(c am)), a ternary complex of P450(cam), reduced putidaredoxin, and d-camphor is formed as an obligatory reaction intermediate. When ligands such as CO, NO, and O-2 bind to the heme iron of P450(cam) in the intermediate complex, the EPR spectrum of reduced putidaredoxin with a characteristic signal at 346 millitesla at 77 K changed into a spectrum having a new signal at 348 m illitesla. The experiment with O-2 was carried out by employing a mutant P4 50(cam) with Asp(251) --> Asn or Gly where the rate of electron transfer fr om putidaredoxin to oxyferrous P450(cam) is considerably reduced. Such a li gand-induced EPR spectral change of putidaredoxin was also shown in situ in Pseudomonas putida, Mutations introduced into the neighborhood of the iron -sulfur cluster of putidaredoxin revealed that a Ser(44) --> Gly mutation m imicked the ligand-induced spectral change of putidaredoxin. Arg(109) and A rg(112), which are in the putative putidaredoxin binding site of P450(cam), were essential for the spectral changes of putidaredoxin in the complex. T hese results indicate that a change in the P450(cam) active site that is th e consequence of an altered spin state is transmitted to putidaredoxin with in the ternary complex and produces a conformational change of the 2Fe-2S a ctive center.