Conformational Changes in Cytochrome P450cam and the Effector Role of Putidaredoxin

Abstract

The cytochromes P450 form an enormous family of over 20 000 enzyme variants found in all branches of life. They catalyze the O2 dependent monooxygenation of a wide range of substrates in reactions important to drug metabolism, biosynthesis and energy utilization. Understanding how they function is important for biomedical science and requires a full description of their notorious propensity for specificity and promiscuity. The bacterial P450cam is an unusual example, having the most well characterized chemical mechanism of all of the forms. It also undergoes an increasingly well characterized structural change upon substrate binding, which may be similar to to that displayed by some, but not all forms of P450. Finally, P450cam is one of the rare forms that have a strict requirement for a particular electron donor, putidaredoxin (pdx). Pdx provides the required electrons for enzyme turnover, but it also induces specific changes in the enzyme to allow enzyme turnover, long known as its effector role. This review summarizes recent crystallographic and double electron–electron resonance studies that have revealed the effects of substrate and pdx binding on the structure of P450cam. We describe an emerging idea for how pdx exerts its effector function by inducing a conformational change in the enzyme. This change then propagates to the active site to enable cleavage of the ferric–hydroperoxy bond during catalysis, and appears to provide a very elegant approach for P450cam to attain both high efficiency and protection from oxidative damage.

Linkage between Proximal and Distal Movements of P450cam Induced by Putidaredoxin

“…Correlation and principal component analysis of MD simulations have been carried out by Carma to determine the correlated motion between residues that may suggest a pathway for allosteric communication in proteins. In a recent review, we reported that helices F and G merge into the same community with helices C, H, and I when Pdx binds to the proximal site of P450cam . In this study, to elucidate the pathway for the Pdx-induced effector function, the center of mass of each residue was considered as a node to calculate a contact map, which was then used to probe the critical side-chain mobility and interactions between helices.…”

“…To clarify this hypothesis, a P450cam mutant, L358P, has been of interest because this mutant introduces an additional movement on the heme pyrrole ring and enhances the donation of an electron from C357 to the heme . However, a previous study of L358P P450cam suggests heme ruffling is less relevant to the Pdx-induced conformational change . Another model for the effector role of Pdx emphasizes the interaction between the C-terminus of Pdx and helix C of P450cam. ,− The camphor-bound P450cam–Pdx crystal structure suggests that helix C of P450cam moves 2–3 Å toward the Pdx binding site, while Pdx W106 adopts a new conformer to maintain its interaction with A113 and N116 of P450cam, ,, which is significant for both the second electron transfer reaction and the binding affinity for P450cam. , However, as the effector role of Pdx is still under debate, the relative movements of elements of the P450cam structure upon Pdx binding in solution require further investigation.…”

Double Electron–Electron Resonance Shows That the Substrate but Not the Inhibitors Causes Disorder in the F/G Loop of CYP119 in Solution

“…Conformational changes in P450 enzymes and their correlations with protein function have been at the center of P450 research for many years. Conformational adaptation occurs in both bacterial − and mammallian P450s , upon binding of substrate, inhibitors, and redox partners. Substrate binding induces large conformational changes in many bacterial P450s.…”

“…Substrate binding induces large conformational changes in many bacterial P450s. P450cam, the most studied and model P450, has been identified in open and closed states. − The largest changes occur in the F/G helices and in the unfolding of helix B′ in P450cam. − P450BM3 was the first to be crystallized in an open state. , Later, several other P450s, such as P450nor and Oxy B, were identified in an open structure before a substrate enters the active site. A thermo-stable P450, CYP119, has been crystallized in three different conformations upon binding of different inhibitors. − Conformational heterogeneity has also been found in some mammalian P450s, such as CYP2C5 .…”