Delicate conformational balance of the redox enzyme cytochrome P450cam

Abstract

The energy landscapes of proteins are highly complex and can be influenced by changes in physical and chemical conditions under which the protein is studied. The redox enzyme cytochrome P450cam undergoes a multistep catalytic cycle wherein two electrons are transferred to the heme group and the enzyme visits several conformational states. Using paramagnetic NMR spectroscopy with a lanthanoid tag, we show that the enzyme bound to its redox partner, putidaredoxin, is in a closed state at ambient temperature in solution. This result contrasts with recent crystal structures of the complex, which suggest that the enzyme opens up when bound to its partner. The closed state supports a model of catalysis in which the substrate is locked in the active site pocket and the enzyme acts as an insulator for the reactive intermediates of the reaction.uring catalysis, an enzyme will traverse a conformational energy landscape that can be highly complex and easily changed by external factors, such as temperature, ionic strength, and crystallization. Cytochromes P450 (CYPs) are b-type hemecontaining monooxygenases found throughout the three domains of life. These enzymes catalyze the regiospecific and stereospecific hydroxylation of various aliphatic and aromatic compounds and are involved in a considerable number of metabolic processes, such as steroid biosynthesis and metabolism of xenobiotics in mammals. The CYP superfamily has been extensively studied over the past five decades, and an understanding of its mechanism is crucial for the development of pharmaceutical compounds that do not inhibit these enzymes. Under other circumstances, it is desirable to inhibit a CYP for therapeutic reasons. For example, compounds that inhibit CYP1B1 and CYP1A2 in humans have been proposed as promising anticancer agents (1). The archetypal member of this superfamily is CYP101A1 (more commonly known as P450cam) from Pseudomonas putida, which was the first CYP for which the 3D structure was determined by X-ray crystallography (2). P450cam catalyzes the hydroxylation of D-camphor to 5-exohydroxycamphor using two electrons, two protons, and molecular oxygen. Putidaredoxin (Pdx) reductase oxidizes NADH and transfers the electrons to Pdx, which, in turn, shuttles electrons to P450cam in two consecutive steps (3-5). This complex reaction is controlled by the enzyme to ensure an efficient coupling between dioxygen reduction and substrate hydroxylation and to avoid side reactions. P450cam must go through a cycle of several conformational and electronic states to perform its catalytic task (6).The first X-ray crystal structures of P450cam showed that the enzyme occupied a closed conformation, both in the presence and absence of substrate; therefore, it was unclear how the substrate was able to bind into the active site (2). Moreover, structures of the intermediates of the catalytic cycle were also in a closed conformation (7), leading to the idea that during the catalytic cycle, P450cam opens to bind camphor, closes to perform hydroxylation, and then re...

Conformational selectivity in cytochrome P450 redox partner interactions

“…For example, Pdx binding shifts P450cam from high to low spin (4), indicating that when Pdx binds, the active site opens up, thus allowing water to enter the active site and coordinate to the heme iron. Also consistent with opening of the active site is that Pdx binding destabilizes the P450cam-oxy complex ∼150-fold (5 (10). That the open form was found in the crystal structures and DEER experiments was dismissed as artifacts of crystallization and the low temperature required for the DEER experiments.…”

Identification of productive and futile encounters in an electron transfer protein complex

“…cytP450cam remains closed on binding to Pdx (30). Low-temperature EPR studies suggested that Pdx binding leads to a mixture of open and closed states for the oxidized cytP450cam but not for the reduced, CO-bound state (31,32).…”

Quantum Mechanics/Molecular Mechanics Modeling of Enzymatic Processes: Caveats and Breakthroughs

“…It is important to bear in mind here ac onformational change to the enzymatic three-dimensional structure is common after substrate binding. [30] Many docking protocols have been developed in order to try and assess the likelihood of varioussubstrate-binding orientations and positions. [29] As can be seen, the major differences in protein folding are observed mostly in the Fa nd Gh elices that form the flexible loop domain [29b] (inside the green rectangle) and it can be hypothesized that substrate binding will closealid of the protein and functions as at rigger to initiate the catalytic cycle.…”