The Flexibility of Carboxylate Ligands in Methane Monooxygenase and Ribonucleotide Reductase: A Density Functional Study Maricel Torrent, Djamaladdin G. Musaev,* and Keiji Morokuma* Cherry L. Emerson Center for Scientific Computation and Department of Chemistry, Emory UniVersity, Atlanta, Georgia 30322 ReceiVed: October 9, 2000 Available experimental data for the active sites of the hydroxylase component of methane monooxygenase (MMOH) and the R2 subunit of ribonucleotide reductase (R2) indicates high flexibility of the ligand environment of the iron centers in these two metalloproteins, suggesting that carboxylate ligands may play a special role for proper enzymatic functioning. By using quantum chemical methods, here we have investigated (1) the so-called 1,2-carboxylate shift (i.e., shift of a bridging carboxylate ligand from µ-1,1 to µ-1,2 between two metal centers), and (2) the monodentate T bidentate rearrangement of terminal carboxylate ligands (bound to only one metal center), in the reduced forms of MMOH and R2. Our results show that (i) MMOH-like and R2-like structures, with a µ-1,1 and µ-1,2 bridged carboxylate ligand, respectively, are energetically very close; (ii) complexes with lower coordination numbers in the Fe 2 center are computed to be slightly more stable than those with higher coordination numbers, and (iii) the two studied carboxylate shifts are easy processes, not only thermodynamically but also kinetically, with activation barriers of only a few kcal/mol. Our conclusion that the carboxylate ligands of dinuclear complexes such as MMOH red and R2 red are very flexible is in a good agreement with the available experimental data. I. Introduction Methane monooxygenase (MMO) and ribonucleotide reduc- tase (RNR) are two of the most extensively characterized members of the binuclear non-heme iron proteins. 1 Several studies of MMOH and R2 show extensive homologies between these two enzymes (for example, both of them contain two similar E/D-X-X-H sequences), as well as flexibility of the ligand environment of the Fe centers. 2-7 In the literature, this ligand flexibility has been postulated to be one of the most important factors for the proper functioning of the enzymes. Indeed, X-ray studies of the core structure of oxidized MMOH (MMOH ox ) isolated from Methylococcus capsulatus show that at 4 °C the two Fe atoms are triply bridged by one hydroxo, and two µ-1,2-carboxylate ligands with an Fe-Fe distance of ∼3.4 Å. 2 Each Fe center has one histidine ligand. In addition, Fe 1 has one terminal aquo and one carboxylate ligand, while Fe 2 has two carboxylate ligands (see Scheme 1). On the other hand, the structure recorded at -160 °C shows an aquo bridge replacing one of the carboxylate bridges; the resulting structure has one µ-OH, one µ-H 2 O, and one carboxylate bridge with a shorter Fe-Fe distance (∼3.1 Å). 3 The fact that crystallographic studies support diamond core structures with short Fe-Fe distances, as well as a structure with a longer Fe-Fe distance, dictated by the nature of one (or more) bridge(s), suggests that the core structure must be relatively flexible. Similarly, the oxidized binuclear active site of the R2 subunit of RNR from Escherichia coli (R2 met ) shows one µ-oxo and one 1,2- carboxylate bridge with an Fe-Fe distance of ∼3.2 Å, and one histidine ligand for each Fe center. 4 In addition, Fe 1 has a terminal aquo and a chelating carboxylate ligand, whereas Fe 2 has two monodentate terminal carboxylate ligands (like in MMOH), and one terminal aquo ligand. As shown in the literature, the oxidized forms of MMOH and R2 (MMOH ox and R2 met ) including two ferric Fe atoms, Fe III , are the resting state of these enzymes. Only their two- electron reduced forms, MMOH red and R2 red , with two ferrous, Fe II , iron centers are capable of reacting with O 2 , the reaction that initiates both the catalytic cycle of MMO and the formation of a stable tyrosyl radical in R2 of RNR. Structural studies demonstrated 2-4,6 that two-electron reduction of MMOH ox and R2 met dramatically changes the ligand environment of the Fe centers. Indeed, for MMOH, during reduction two hydroxo/aquo bridging ligands move out and one of the carboxylate ligands of Fe 2 , Glu243, shifts to form a monodentate bridge between the two metals as well as coordinating to Fe 2 center in a bidentate manner. 3 Therefore, the two Fe atoms are changed from six-coordinate (6C) to five-coordinate (5C), with one vacant site for each Fe. Spectroscopic studies are in accord with such a (5C, 5C) assignment for the reduced state of MMOH. 5 Similarly, in R2 met the two-electron reduction leads to dissocia- tion of two oxo/aquo bridging ligands. 6 However, upon reduction of R2 met the terminal ligand Asp84 shifts from chelating to monodentate terminal position to Fe 1 , a shift which has no analogue in the counterpart reduction of MMOH. Carboxylate ligand Glu238 in R2, which moves from terminal monodentate in Fe 2 to a bridging position, also gives a bidentate bridge rather than monodentate as in MMOH. Thus, according to crystal- lographic studies, 6 R2 red has two approximately equivalent 4C Fe centers. Spectroscopic data, 7 on the contrary, indicate that Fe 1 and Fe 2 atoms in R2 red are 5C and 4C, respectively. The challenge comes in reconciling the CD/MCD studies with the structural data. Here, we have preferred to chose a starting structural model for R2 with inequivalent sites (Scheme 1). So far, the asymmetric view seems to have a larger acceptance. This view has been supported by a very recent study 8 proposing that Glu204 is actually bidentate to Fe 2 in solution, and therefore, suggesting that Fe 2 is a 5C site. Further support to the asymmetric view comes from the reduced binuclear active site 322 J. Phys. Chem. B 2001, 105, 322-327 10.1021/jp003692m CCC: $20.00 © 2001 American Chemical Society Published on Web 12/07/2000