Novel routes to catalytic intermediates in the cytochrome P450 catalytic cycle

The proteins known as cytochromes P450 (P450s) are essential in physiology of all life forms. They are heme-binding proteins, and bind the same heme cofactor as does the oxygen carrying blood protein hemoglobin. Like hemoglobin, P450s also bind molecular oxygen (O2). However, unlike hemoglobin they reduce bound oxygen with electrons delivered to the heme from partner proteins, and which ultimately are derived from the cell coenzyme NADPH. This enables P450s to split the oxygen molecule into its component atoms. One of the two atoms is used to form water (H2O), while the other is used to oxygenate an organic substrate molecule bound by the P450 close to its heme iron. Frequently, hydroxylation (introduction of an OH group) is catalysed. In humans, activity of P450s is essential for production of steroid hormones, and also for creation of many lipid molecules essential for signalling within the body (e.g. for activation of the immune system). However, humans have 57 different P450s, and their most famous roles are in detoxification and removal of drugs and other xenobiotics from the body / performed mainly by hepatic P450s. In bacteria and lower eukaryotes, the P450s have important roles in pathways that allow unusual molecules (e.g. camphor) to be used to provide energy for growth, and are essential for production of molecules such as antibiotics (e.g. erythromycin). The ability of P450 enzymes to introduce oxygen atoms at defined positions in organic molecules has also attracted much attention from organic chemists, who are looking for cleaner and more environmentally friendly routes to synthesis of drugs and other important molecules. A fundamental understanding of P450 structure and activity is essential to understand how they achieve their biological functions, and how they can be applied for biotechnological roles. Also, there is enormous interest in understanding how prescribed drugs bind to individual P450s (and how molecules of biotechnological interest bind to the relevant P450s), since this can lead to accurate predictions of how individual P450s act on these molecules, their lifetimes in the body and how these parameters can be changed by altering the drug structure. The usual way of determining binding modes of substrates/drugs to P450s is to form crystals of the complex made between the P450 and the drug, and then use the technique of x-ray diffraction to obtain the crystal structure. In this proposal, we seek to address fundamental questions relating to how P450s 'activate' oxygen and catalyse hydroxylation reactions. Specifically, we will use modern kinetic techniques (including laser flash photolysis) to provide evidence for formation of transient reactive heme species that are considered critical for oxygenation chemistry. Also, we will use these methods to answer a critical question relating to whether two different reactive species are formed in the P450 reaction 'cycle' and if these have differing types of activities that could be exploited biotechnologically. In addition, we will address serious issues relating to the relevance of binding modes seen for substrates in different P450 x-ray structures. We will use a model system (P450 BM3) to establish whether an observed substrate binding mode is relevant to catalysis in the P450 and to challenge hypotheses suggesting that the substrate re-positions as the P450 is reduced, or whether thermal effects are critical for causing substrate to relocate. Collectively, this work will answer fundamental questions on the nature of P450 catalysis and the relevance of distinct reactive intermediates in the process. Also, it will define the relevance of substrate binding mode and substrate relocation in a key model P450, with important ramifications for rationalising how substrates bind to biomedically relevant P450s. Thus, the study proposed has wide ranging relevance to understanding P450 activity in mammalian physiology and for biotechnological applications.

P450s oxygenases have many roles in mammalian physiology/drug metabolism, and enormous biotechnological potential in regio- and stereo-selective oxygenation of organic molecules. Transient nature of intermediates in the P450 cycle has meant that a key species essential for P450-dependent reactions (ferryl-oxo compound 1) has not been isolated, and even a precursor complex (ferric hydroperoxy compound 0) has only been seen by low temp. 'annealing' of a precursor. Isolation of these complexes is important, due to controversy on relevance of compound 0, postulated to catalyse 'soft' oxidations (e.g. C=C bonds) by experimentalists, but contradicted by computational biologists who suggest compound 1 has two states of differing reactivity. Using laser methods we've developed (photolysis of NAD[P]H to reduce P450 heme in microseconds) we will study resting P450 BM3 (a model P450) and intermediates arrested in mutant forms to obtain data on properties of intermediates and affinity for ligands (e.g. dioxygen). By distinct approaches, we will use laser methods to isolate compounds 0 and 1 and determine their features. We will examine compound 0 properties to address its oxidant capacity. In parallel, we will exploit our successes in isolating high resolution and reduced BM3 crystal forms to address key questions on relevance of a substrate binding mode distant from the heme. We will solve structures of substrate complexes in oxidised/reduced P450s, and at different temperatures to challenge theories that heme reduction and/or thermal effects induce substrate relocation to a catalytically relevant site. We will also solve structures for the related CYP102A3, that lacks a substrate binding motif we consider promotes binding of BM3 substrate in a catalytically irrelevant mode. Data will have fundamental impact on understanding P450 catalysis and relevance of intermediates, and on mechanism of a model P450, as well as introducing new techniques for transient kinetic studies.