Linking of cytochrome P450_cam and putidaredoxin by a co-ordination bridge

Abstract

Cytochrome P450_cam (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 other studies of this system, in that strong binding of PdX to P450 is crucial for good coupling and for release of 5-exo-hydroxycamphor.

• Cytochrome P450
• ferredoxin
• chemical modification
• peptide mapping
• coupling
• hydroxylation

Notes

1. The cytochrome P450cam system uses NADH, but the adrenal class I P405 systems and the class II systems use NADPH as final reductant.

2. This procedure is dangerous; care should be taken not to let peroxides accumulate in the still and when quenching the sodium.

3. NiSO₄ was dissolved in water.

4. RuCl₃ was dissolved in ethanol.