Quantum Chemical Studies of Methane Monooxygenase: Comparision with P450 Victor Guallar*, Benjamin F Gherman*, Stephen J Lippard† and Richard a Friesner*
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236 Quantum chemical studies of methane monooxygenase: comparision with P450 Victor Guallar*, Benjamin F Gherman*, Stephen J Lippard† and Richard A Friesner* The catalytic pathways of soluble methane monooxygenase the system for which the greatest amount of kinetic and (sMMO) and cytochrome P450CAM, iron-containing enzymes, high-resolution structural data are presently available. are described and compared. Recent extensive density functional ab initio electronic structure calculations have Both the hydroxylase component (MMOH) of sMMO and revealed many similarities in a number of the key catalytic cP450 employ iron as the transition metal component at steps, as well as some important differences. A particularly the enzyme active site. MMOH is a non-heme diiron interesting and significant contrast is the role played by the protein [5,12,26], whereas cP450 employs a heme iron protein in each system. For sMMO, the protein stabilizes [27,28] as its catalytic agent. Figure 1 displays the active various species in the catalytic cycle through a series of site structures of the resting forms for both enzymes as carboxylate shifts. This process is adequately described by a determined by X-ray crystallography. Despite considerable relatively compact model of the active site (~100 atoms), differences in these core structures, the transition metal providing a reasonable description of the energetics of functions similarly in both enzymes, as discussed below. hydrogen atom abstraction. For P450CAM, in contrast, the inclusion of the full protein is necessary for an accurate The catalytic cycles in both MMOH and cP450 are similar, description of the hydrogen atom abstraction. although key details differ significantly. We outline common elements of the catalytic cycle as follows: Addresses *Department of Chemistry, Columbia University, New York 10027, USA 1. The resting state is activated by injecting electrons to †Department of Chemistry, Massachusetts Institute of Technology, reduce the iron atom(s) so it (or they) can effectively bind Cambridge, Massachusetts 02139, USA dioxygen. A partner protein capable of long-range electron Current Opinion in Chemical Biology 2002, 6:236–242 transfer (MMO reductase [29], and putidaredoxin reductase for cP450 [30–32]) is required. 1367-5931/02/$ — see front matter © 2002 Elsevier Science Ltd. All rights reserved. 2. Dioxygen is bound and reduced, displacing a weakly Abbreviations bound water molecule. Reductive activation of dioxygen cP450 cytochrome P450 DFT density functional theory results in a change of spin and redox state of the iron atom(s) MMOH methane monooxygenase hydroxylase and a substantial conformational change in MMOH. QM/MM quantum mechanics/molecular mechanics sMMO soluble methane monooxygenase 3. The enzyme converts to form the active species, in which one oxygen atom of the dioxygen is prepared to Introduction abstract a hydrogen atom from the substrate. Conversion of a C–H bond into an alcohol, by formal insertion of an oxygen atom from dioxygen, is a common 4. The active species, containing a highly reactive oxygen and important biochemical reaction [1,2]. Activation of the atom (oxene) attached to one or more Fe(IV) atoms, C–H bond at room temperature is a challenging problem, abstracts a hydrogen from the substrate via a linear particularly when specificity is desired with regard to C⋅⋅⋅H⋅⋅⋅O transition state. Rotation of the resulting –OH reactants and products, as in a biochemical context. A moiety created at the catalytic core then allows attack by a highly reactive oxidant is required, yet this species must substrate carbon leading to the alcohol, which rapidly perform only the requisite C–H conversion to the alcohol. detaches from the core as the product. This process can proceed either through a bound radical or by a concerted To solve this problem, nature has principally relied upon reaction in which this species does not form. transition-metal-containing enzymes that exhibit complex catalytic cycles. We consider two such enzymes in this 5. The resting state is regenerated. review: soluble methane monooxygenase (sMMO) and cytochrome P450 (cP450). sMMO catalyzes the conversion Figure 2 describes these various steps for the MMOH and of methane, NADH and dioxygen to methanol and water cP450 catalytic cycles. [3–7,8••,9••,10–12]. A variety of cP450 enzymes have been the subject of experimental investigation [13–18]. Here, Computational methods we focus specifically on the bacterial enzyme cytochrome Although much has been learned from experimental P450cam, [19••,20••,21–25] named for its preferred natural studies of both MMOH and cP450, a detailed structural substrate, camphor. cP450cam was selected because it is and energetic description of the reaction mechanisms is Quantum chemical studies of methane monooxygenase: comparision with P450 Guallar et al. 237 Figure 1 Geometries from X-ray crystal structures of the core of the active sites of the resting MMO forms of both enzymes. Fe Fe Camphor P450cam Thr252 Water channel Fe Current Opinion in Chemical Biology difficult, if not impossible, to formulate, without the use of can provide excellent accuracy for relative energetics of computational methods. Advances over the past decade in transition-metal-containing systems. Using Jaguar, it is possible computational chemistry models and algorithms, as well as to include on the order of 150 atoms at a QM level with computer hardware, have rendered tractable the treatment relatively modest computational effort. QM/MM methods of the reaction chemistry of transition-metal-containing allow a QM region of this size to be embedded in an MM enzymes. The studies reviewed here employ ab initio representation of the entire protein. Our expectation is that quantum chemical method based on density functional the energies of species computed by these methods will have theory (DFT) [33,34], as well as mixed quantum mechanics/ an accuracy of ~3–5 kcal mol–1, or better in favorable cases. molecular mechanics (QM/MM) [35,36] methods in the This degree of accuracy is sufficient for evaluating the case of cP450. The Jaguar suite (all quantum chemistry relative energies of intermediates and transition states in the calculations described below were carried out with cP450 and MMOH catalytic cycles, affording a qualitative Jaguar 4.1, Schrödinger, Inc., Portland, OR, 1991–2000) of understanding of the reaction pathways and allowing one to ab initio quantum chemistry programs provide a particularly compare the two enzymes under study. efficient platform for the treatment of large transition- metal-containing systems, as documented extensively in For MMOH, our results are based on a QM model of ~100 previous publications [3,4]. atoms, which contains key protein hydrogen bonds that enforce structural stability at the core region; our model for DFT methods, when used with large basis sets and a studying the hydrogen atom abstraction step is shown in reasonable (if approximate) treatment of open-shell systems, Figure 3. The compact, globular nature of the diiron core 238 Guest section: computational bioinorganic chemistry II Figure 2 Hred Hperoxo +O2 –H2O +2e– +2H+ –H2O +R CH +H2O 3 –R3COH R3COH Hox Oxygenated substrate Q –1 +Substrate +e– +e– + +O2 +2H –H2O –H2O Ferric resting state Oxyferrous Oxyferryl compound I Oxygenated substrate Current Opinion in Chemical Biology Various steps for the MMOH and cP450 catalytic cycles. makes it possible to obtain meaningful results without of the methodology. Structures of the reduced and oxidized including the rest of the protein. For cP450, it is necessary enzyme have been determined for MMOH by one of our to use QM/MM methods if a quantitative treatment of the research groups [37,38]; our calculations reproduce these catalytic cycle is desired. The heme group, substrate, and structures to within ~0.5 Å, approaching the experimental key catalytic waters and protein side-chains form a loosely resolution. For cP450, we make extensive use of the recently aggregated, delocalized assembly that would disassemble determined structures [19••] that provide the first information without the protein to hold the various pieces in place. about transient intermediates important in that system. Moreover, the specific geometries at which these compo- nents are held are critical for function. The use of QM/MM Results methods obviates this problem and allows a quantitative We focus here on steps 3 and 4 of the catalytic process, for assessment of electrostatic stabilization and strain energy which we have produced extensive results that allow the imposed by the protein, which is crucial to its catalytic mechanisms for MMOH and cP450 to be contrasted in an function. The QM region, comprising the groups men- interesting fashion. Step 1 is difficult to model quantita- tioned above, is ~130 atoms in the studies described below. tively because of the participation of partner proteins. Experimental structures of the pertinent protein–protein For both systems, we employ high-resolution X-ray crystal complexes are not yet available, although the MMOH- structures as a starting point for model building and calibration binding surfaces have been identified for MMOB and Quantum chemical studies of methane monooxygenase: comparision with P450 Guallar et al. 239 MMOR [39–41]. We have verified that step 2 is thermo- Figure 3 dynamically favorable in both systems, but quantitative characterization of the processes is still ongoing. Finally, we have not yet investigated step 5. Preparation of