Structure of camphor bound T260A mutant of CYP101D1

Abstract

Cytochromes P450 catalyze a variety of monooxygenase reactions that require electron transfer from redox partners. P450cam, a soluble bacterial cytochrome from Pseudomonas putida that hydroxylates camphor, has served as the primary model to address questions on the detailed mechanisms of electron transfer and O2 activation. The entire P450cam system consists of an iron-sulfur ferredoxin, putdiaredoxin (Pdx), that transfers electrons from the FAD protein, putidaredoxin reductase (Pdr), to P450cam. P450cam is unusual in having a strict and specific requirement for Pdx as an electron donor, which suggests that the specificity for Pdx is related to the Pdx-induced structural change required for oxygen activation.1–3 The recent P450cam-Pdx crystal and NMR structures4,5 indicate that the effector role of Pdx is to shift P450cam toward the open conformation, which enables the establishment of a water-mediated H-bonded network that is required for proton-coupled electron transfer and oxygen activation.5 Pdx binding has been proposed to free the critical residue, Asp251, from salt bridging interactions with Arg186 and Lys178 in order to serve its catalytic function6,7 in shuttling protons to dioxygen. CYP101D1 is a close homolog to P450cam and catalyzes exactly the same camphor hydroxylation reaction, uses a similar ferredoxin (Arx) and ferredoxin reductase (Adr), but exhibits subtle and possibly important differences.8 Adjacent to the heme iron ligand, Cys357, is Leu358 in P450cam while this residue is Ala in CYP101D1. Leu358 plays a role in binding of the P450cam redox partner, putidaredoxin (Pdx).9 On the opposite side of the heme about 15 – 20 A away the critical residue Asp251 in P450cam forms strong ion pairs with Arg186 and Lys178. In CYP101D1 a Gly replaces Lys178. Thus, the local electrostatic environment and ion pairing is substantially different in CYP101D1. Another important difference relates to the effect of K+ ions on substrate binding and spin state. Potassium ions promote both susbtrate binding and high-spin P450cam.10 P450cam has a K+ binding site near Tyr96 whose side chain OH group H-bonds with the camphor carbonyl oxygen of camphor so K+ helps to stabilize key substrate-protein interactions.11 CYP101D1 lacks this K+ binding site and K+ has only a modest effect on the fraction of high-spin CYP101D1.8 The addition of 200 mM KCl to the CYP101D1-camphor complex increases the spin state from 30% to only 40%.8 In our recent study aimed at comparing these two enzymes, these sites were systematically mutated in P450cam to the corresponding residues in CYP101D1.12 The data show that although individually the mutants have little effect on activity or structure, in combination there is a major drop in enzyme activity due the mutants being locked in the low-spin state thus preventing electron transfer from the P450cam redox partner, Pdx. Overall these results illustrate the delicate balance between the open and closed conformational states13 as well as a strong structural connection between the Pdx binding site (Leu358) and the region around Asp251 that undergoes a large structural change in the open/closed transition in P450cam. The results along with some structural and biochemical studies on CYP101D1 indicate that these complex interactions between spin-state, redox partner binding, and conformational changes might be less stringent or different in CYP101D1 primarily because this P450 is predominantly low-spin even in the presence of substrate14 but still is as active as P450cam. This opens up some interesting questions on how two such similar P450s can behave so differently and what, if any, the connection is between these various levels of conformational dynamics, activity, and biological function. Here we present the crystal structures of cyanide and camphor bound complexes of wild type CYP101D1 and its active site mutants, D259N and T260A, and enzyme activities. Since CYP101D1 is so similar to P450cam, a comparison of the two with respect to redox partner specificity has also been addressed to better understand whether the effector/allosteric role of redox partner binding is a general property of P450s or is unique to P450cam.