Computational Studies of Reaction Mechanisms of Methane Monooxygenase and Ribonucleotide Reductase MARICEL TORRENT, 1 DJAMALADDIN G. MUSAEV, 1 HAROLD BASCH, 1,2 KEIJI MOROKUMA 1 1 Contribution from the Cherry L. Emerson Center for Scientific Computation and Department of Chemistry, Emory University, Atlanta, Georgia 30322 2 Department of Chemistry, Bar Ilan University, Ramat Gan 52900, Israel Received 14 February 2001; Accepted 16 July 2001 Abstract: An overview of the computational efforts made by our group during the last few years in the field of nonheme diiron proteins is presented. Through application of ab initio methodology to a reasonable set of molecular models, significant progress is made in understanding how the soluble Methane Monooxygenase system achieves the hydroxylation of methane and how the catalytic cycle of Ribonucleotide Reductase is initiated. In particular, the current studies reveal in more detail (1) the nature of key intermediates in the reaction cycles of these two metalloenzymes, (2) details of how the iron centers regulate the systems, and (3) important aspects of how the carboxylate ligands in the active sites may tailor the enzymatic needs of the metalloprotein. This knowledge also leads to novel connections between the two enzymes. The coordinative unsaturation and carboxylate shifts investigated herein are two properties that are likely to be of more general impact in nonheme proteins. The control of the redox chemistry of the enzyme by the binuclear metal center, also analyzed here, should find common ground among other bimetallic systems as well. © 2002 John Wiley & Sons, Inc. J Comput Chem 23: 59–76, 2002 Key words: nonheme protein chemistry; diiron enzymes; bond activation; reaction mechanisms; molecular modeling Introduction Metalloenzymes with nonheme diiron centers in which the iron ions are bridged by an oxide (or a hydroxide) and carboxylates (glutamate or aspartate) have emerged as an important class of enzymes in the past 2 decades. 1 The crystal structures of several members of this class isolated from mammals, plants or bacteria, are now available. The first structurally characterized protein of the binuclear non- heme family was hemerythrin (Hr), 1a the O 2 carrier protein in marine invertebrates (Scheme 1, top). To the best of our knowl- edge, this is the only example of a diiron protein involved in oxygen transport. In contrast, this type of metal center has been frequently employed by living organisms for dioxygen activation and substrate oxidation. Well-known examples are the iron centers in methane monooxygenase (MMO) from methanotrophic bacteria, ribonucleotide reductase (RNR) from Escherichia coli and eukary- otes, plant desaturases, and a variety of bacterial monooxygenases. Among the members of the binuclear nonheme protein family, MMO and RNR can be regarded as “twin” enzymes. The overall homology is very weak, but experimental studies 2 have unveiled extensive similarity of the active sites of MMO and RNR, which makes them distinct from other members of the same class, as it is clear from a comparison of the structures of the three enzymes shown in Scheme 1. The active site of Hr consists of a triply bridged diiron core that is coordinated by five terminal His ligands. MMO and RNR, on the other hand, have very similar active sites, which are carboxylate rich with respect to that of Hr. The two ferrous iron centers are bridged by two carboxylate ligands; 3 each Fe center has one histidine and one terminal oxo-ligand. In addition, one of the irons (Fe 1 ) includes an additional water molecule. Moreover, both MMO and RNR contain two Glu/Asp-X-X-His sequences each. Several experimental studies 3 have shown that the resting state of MMO and RNR is the oxidized form of these enzymes (MMO ox and RNR met ), which includes two ferric Fe atoms, Fe III . Only the reduced forms, MMO red and RNR red , with two ferrous, Fe II , iron centers are capable of reacting with O 2 , the reaction that initiates both catalytic cycles. Such differences in structure between Hr and MMO/RNR somehow affect the function of these three enzymes. For example, in the former the O 2 molecule is not activated as in MMO and RNR. In Hr, O 2 is simply transported and, therefore, re- versibly bound to the enzyme (which undergoes minimal changes). In MMO and RNR, the O—O bond is irreversibly cleaved. Despite the observed structural similarities of their active sites, MMO and RNR perform different catalytic functions that involve Correspondence to: K. Morokuma, e-mail: morokuma@emory.edu; or D. G. Musaev, e-mail: dmusaev@emory.edu Contract/grant sponsors: Spanish Ministerio de Educación y Cultura (postdoctoral fellowship to M.T.), the Emerson Center (visiting fellowship to H.B.) Contract/grant sponsor: the National Science Foundation; contract/grant number: CHE-9627775 © 2002 John Wiley & Sons, Inc.