Novel routes to catalytic intermediates in the cytochrome P450 catalytic cycle

Lead Research Organisation: University of Manchester
Department Name: Life Sciences

Abstract

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.

Technical Summary

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.

Publications

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Guengerich FP (2013) Unusual cytochrome p450 enzymes and reactions. in The Journal of biological chemistry

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McLean K (2015) Cytochrome P450

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McLean KJ (2015) Biological diversity of cytochrome P450 redox partner systems. in Advances in experimental medicine and biology

 
Description The cytochromes P450 (P450s) are heme containing enzymes that bind to atmospheric (molecular) oxygen (O2), and then split the oxygen into its component atoms - making a molecule of water (H2O) with one of the oxygen atoms, and inserting the other atom of oxygen into a substrate molecule that binds close to the O2-bound heme. Frequently this results in hydroxylation of the substrate. This is the reaction catalysed by the P450 BM3 enzyme from the soil bacterium Bacillus megaterium, which is the fastest known P450 hydroxylase and the subject of studies done in this project. BM3 is a P450 that is naturally fused to its efficient, electron donating partner, a factor underpinning its high levels of activity.
The aim of this research was to use the biotechnologically important BM3 enzyme as a model system for investigating the catalytic properties of P450s and to probe for formation of reactive oxygen-bound intermediates that are involved in the catalytic cycle of the BM3 P450, and which ultimately result in oxidation of the substrate. This research used a combination of methods including computational and experimental techniques (the latter including fast reaction methods of stopped-flow absorption spectroscopy and laser flash photolysis); and employed mutagenesis methods to alter the properties of the P450 in order to stabilize certain oxygen bound species.
Research performed furthered knowledge of structure/mechanism in a biotechnologically important P450 enzyme. Most significant achievements were (i) The generation of mutants of P450 BM3 that stabilize its ferrous-oxy complex and/or alter its heme iron coordination state. The former mutants enabled analysis of binding kinetics for gaseous ligands (oxygen, CO) to the heme iron and allowed studies to probe formation of later catalytic cycle intermediates; (ii) Development of methods to rapidly reduce P450s using laser excitation and to form (using peroxynitrite or peroxide surrogates) reactive iron-oxo intermediates. Limited evidence (low yields) for formation of the most reactive species (the iron-oxo intermediates referred to as compound 0 and compound I) was obtained through fast reaction studies, but computational work favoured strongly the catalytic relevance of only the very short-lived compound I intermediate. During the course of this study, research was done elsewhere (using a thermostable P450 operating at ambient temperature) that provided convincing data for formation of compound I and its catalytic potency. Near the end of this project, ourselves and others were also able to get good spectroscopic evidence for compound I formation in a different P450 enzyme in which catalysis is naturally driven by hydrogen peroxide. Through enzymatic, structural and computational studies, novel data were obtained for the mechanism of inter-monomer electron transfer in the P450 BM3 enzyme, which we showed naturally to be a dimeric enzyme. In addition, the dominant role of the ferryl-oxo (compound I) intermediate (over the ferric-hydroperoxo compound 0) in BM3 substrate oxidations was shown computationally. Other mechanistic and modelling studies probing the BM3 mechanism indicated that substrate (fatty acid) migration from a non-catalytically competent state to a position proximal to the heme occurs, and that this process is likely not related to changes in the reduction or oxygen binding state of the P450. Several P450 BM3 mutants were made in the course of this project, and in some cases we were able to show considerable changes in the way the heme iron was bound (coordinated) by protein amino acids. This allowed us to identify a number of heme iron coordination states that had not previously been observed in nature, and to identify novel conformational states occupied by the P450. Collectively, a large body of novel data on the catalytic and mechanistic features of a biotechnologically important P450 enzyme were collected, and new insights were made into how conformational changes occur and how these may be related to the progression of the P450 catalytic cycle.
Exploitation Route Important data were collected in relation to conformational states occupied by the target P450, and further studies here (including dynamics analysis) could enable understanding of how conformational rearrangements are related to the catalytic process of P450s. Novel ligation modes of heme iron were also identified and these provide templates for identifying proteins in which these occur naturally. Methods developed here for rapid P450 reduction should be used to explore reactions of heme (and other redox cofactor) binding proteins with oxygen and other substrates in order to determine mechanistic details in P450s and in many other important redox enzymes.
Sectors Chemicals,Pharmaceuticals and Medical Biotechnology

 
Description The project involved studies of structure, mechanism and fast reaction chemistry in a biotechnologically important cytochrome P450 enzyme. Methods were developed (particularly by laser flash excitation) for rapid reduction of the P450 heme iron in the P450 BM3 enzyme and to study its reactions with oxygen. These methods continue to be used in our research, and can find applications in studies of several other redox enzymes in order to explore their mechanism and in efforts to identify transient reactive species. Protein engineering studies done in the course of this project have provided a range of BM3 mutants with novel properties (including altered substrate binding, novel heme iron ligation modes and perturbations to heme iron potential) and we have exploited a number of these variants for further studies into transient P450 cycle intermediates and for uses in synthetic biology applications, including production of drug and steroid metabolites.
First Year Of Impact 2012
Sector Chemicals,Pharmaceuticals and Medical Biotechnology
Impact Types Societal

 
Description Manchester Institute of Biotechnology Open Day - annual event from 2012 onwards 
Form Of Engagement Activity Participation in an open day or visit at my research institution
Part Of Official Scheme? No
Geographic Reach Regional
Primary Audience Schools
Results and Impact Scientific demonstrations to senior secondary school students to enthuse them about a scientific career and to provide advice on career development and the courses on offer at the University of Manchester.

Annual event - such that lessons are learned from one year's activity and are carried forward to the following year's presentations.
Year(s) Of Engagement Activity 2012,2013,2014,2015
 
Description Schools visit (Wilmslow) 
Form Of Engagement Activity A talk or presentation
Part Of Official Scheme? No
Geographic Reach Local
Primary Audience Schools
Results and Impact Presentation to primary school children in final year on general science/genetics - talk sparked questions and general discussion

Students registered interests in scientific career. Invite for further talk in following year obtained.
Year(s) Of Engagement Activity 2007,2008,2009,2010,2011