Linking of cytochrome P450camand putidaredoxin by a co-ordination bridge

Cytochrome P450cam (CYP101) catalyzes the oxidation of D(+)-camphor at the 5 position. The enzyme couples the reduction of dioxygen to the oxidation of the substrate. To transfer electrons from the reductant (NADH) to the cytochrome, two additional proteins are required. These are putidaredoxin (PdX) and putidaredoxin reductase (PdR). We have chemically linked a form of PdX with a histidine tag at the C-terminus to the P450. To accomplish this, we have modified cysteine 334 on P450 with a bipyridinyl group, and co-ordinated the C-terminal histidine tag of PdX by the addition of Ni2+ or Ru3+. The Ru3+ complex was the most stable. The non-linked system gave mostly 5-ketocamphor, a product of two consecutive hydroxylations, and H2O2, a product of 2-electron uncoupling. The Ni2+ complex gave both 5-exo-hydroxycamphor and 5-ketocamphor, but it also uncoupled. The Ru3+ complex gave a single product (5-exo-hydroxycamphor) and did not uncouple at the optimal PdR concentration. Our results are consistent with othe...