Redox partner recognition and selectivity of cytochrome P450lin (CYP111A1).

The strict requirement of cytochrome P450cam for its native ferredoxin redox partner, putidaredoxin (Pdx), is not exhibited by any other known cytochrome P450 (CYP) system and the molecular details of redox partner selectivity are still not completely understood. We therefore examined the selectivity of a related Pseudomonas cytochrome P450, P450lin, by testing its activity with non-native redox partners. We found that P450lin could utilize Arx, the native redox partner of CYP101D1, to enable turnover of its substrate, linalool, while Pdx showed limited activity. Arx exhibited a higher sequence similarity to P450lins native redox partner, linredoxin (Ldx) than Pdx, including several residues that are believed to be at the interface of the two proteins, based on the P450cam-Pdx complex structure. We therefore mutated Pdx to resemble Ldx and Arx and found that a double mutant, D38L/∆106, displayed higher activity than Arx. In addition, Pdx D38L/∆106 does not induce a low-spin shift in linalool bound P450lin but does destabilize the P450lin-oxycomplex. Together our results suggest that P450lin and its redox partners may form a similar interface to P450cam-Pdx, but the interactions that allow for productive turnover are different.