Derivatives of vitamin B12 are used in methyl group transfer in biological processes as diverse as methionine synthesis in humans and CO2 fixation in acetogenic bacteria1–3. This seemingly straightforward reaction requires large, multimodular enzyme complexes that adopt multiple conformations to alternately activate, protect, and perform catalysis on the reactive B12 cofactor. Crystal structures determined thus far have provided structural information for only fragments of these complexes4–12, inspiring speculation regarding the overall protein assembly and conformational movements inherent to activity. Here we present X-ray crystal structures of a complete ~220 kDa complex that contains all enzymes responsible for B12-dependent methyltransfer, namely the corrinoid iron-sulfur protein (CFeSP) and its methyltransferase (MeTr) from the model acetogen Moorella thermoacetica. These structures provide the first three-dimensional depiction of all protein modules required for the activation, protection, and catalytic steps of B12-dependent methyltransfer. In addition, the structures capture B12 at multiple locations between its “resting” and catalytic positions, allowing visualisation of the dramatic protein rearrangements that enable methyltransfer and identification of the trajectory for B12 movement within the large enzyme scaffold. The structures are also presented alongside in crystallo UV-vis spectroscopic data, which confirm enzymatic activity within crystals and demonstrate the largest known conformational movements of proteins in a crystalline state. Taken together, this work provides a model for the molecular juggling that accompanies turnover and helps explain why such an elaborate protein framework is required for such a simple, yet biologically essential reaction.

Acetyl-CoA synthase (ACS) catalyzes the synthesis of acetyl-CoA from CO, coenzyme A (CoA), and a methyl-group from the CH 3 -Co 3+ site in the corrinoid iron-sulfur protein (CFeSP). These are the key steps in the Wood-Ljungdahl pathway of anaerobic CO and CO 2 fixation. The active site of ACS is the A-cluster, which is an unusual nickel-iron-sulfur cluster. There is significant evidence for the catalytic intermediacy of a CO-bound paramagnetic Ni species, with an electronic configuration of [Fe 4 1+ species (A red *) with a rhombic electron paramagnetic resonance spectrum (g-values of 2.56, 2.10, 2.01) and an extremely low (1 kJ/mol) barrier for recombination with CO. We suggest that the photolytically generated A red * species is (or is similar to) the Ni p 1+ species that binds CO (to form the Ni p 1+ -CO species) and the methyl group (to form Ni p -CH 3 ) in the ACS catalytic mechanism. The results provide support for a binding site (an "alcove") for CO near Ni p , indicated by X-ray crystallographic studies of the Xe-incubated enzyme. We propose that, during catalysis, a resting Ni p 2+ state predominates over the active Ni p 1+ species (A red *) that is trapped by the coupling of a one-electron transfer step to the binding of CO, which pulls the equilibrium toward Ni p 1+ -CO formation. KeywordsCO dehydrogenase; acetyl-CoA synthase; photolysis; nickel; EPR spectroscopy; enzyme kinetics; iron-sulfur clusterCarbon monoxide dehydrogenase/acetyl-CoA synthase (CODH/ACS) catalyzes the key steps in the Wood-Ljungdahl pathway of anaerobic CO 2 fixation, which provides carbon and energy for a variety of anaerobic microbes (1)(2)(3)(4) central CODH subunits, each of which is attached to an ACS subunit, forming an α 2 β 2 complex (5,6). In the CODH subunit, CO 2 is reduced to CO, which then travels through a 70 Å tunnel to the A-cluster of ACS (7), Here, CO, a methyl-group from the CH 3 -Co 3+ cofactor in the corrinoid iron-sulfur protein (CFeSP) and coenzyme A (CoA) (1) are converted to acetyl-CoA. Based on the results of X-ray crystallographic (5,8) and biochemical (9,10) studies, the active A-cluster is known to be composed of a [Fe 4 S 4 ] cluster bridged through cysteine to the proximal Ni (Ni p ) of a dinuclear Ni center (Figure 1), an arrangement similar to the Fe-only hydrogenases in which a [Fe 4 S 4 ] cluster and a binuclear Fe site are bridged by a Cys residue (11,12). The substrate binding site is ambiguous; however, computational results (13) combined with biochemical and spectroscopic experiments (10,(14)(15)(16)) and studies of model complexes (17,18) suggest that Ni p is the binding site. Therefore, the purpose of discussion, we will refer to the CO complex as involving Ni p -CO, as shown in the mechanism in Figure 2. Ni p changes ligation and oxidation states during catalysis, whereas the distal Ni (Ni d ), which is ligated by two deprotonated amides and two cysteine thiolates in a Cys-Gly-Cys motif, appears to remain square planar in the +2 oxidation state (17). Various details of the mecha...

Carbon dioxide and carbon monoxide are important components of the carbon cycle. Major research efforts are underway to develop better technologies to utilize the abundant greenhouse gas, CO2, for harnessing ‘green’ energy and producing biofuels. One strategy is to convert CO2 into CO, which has been valued for many years as a synthetic feedstock for major industrial processes. Living organisms are masters of CO2 and CO chemistry and, here, we review the elegant ways that metalloenzymes catalyze reactions involving these simple compounds. After describing the chemical and physical properties of CO and CO2, we shift focus to the enzymes and the metal clusters in their active sites that catalyze transformations of these two molecules. We cover how the metal centers on CO dehydrogenase catalyze the interconversion of CO and CO2 and how pyruvate oxidoreductase, which contains thiamin pyrophosphate and multiple Fe4S4 clusters, catalyzes the addition and elimination of CO2 during intermediary metabolism. We also describe how the nickel center at the active site of acetyl-CoA synthase utilizes CO to generate the central metabolite, acetyl-CoA, as part of the Wood-Ljungdahl pathway, and how CO is channelled from the CO dehydrogenase to the acetyl-CoA synthase active site. We cover how the corrinoid iron–sulfur protein interacts with acetyl-CoA synthase. This protein uses vitamin B12 and a Fe4S4 cluster to catalyze a key methyltransferase reaction involving an organometallic methyl-Co3+ intermediate. Studies of CO and CO2 enzymology are of practical significance, and offer fundamental insights into important biochemical reactions involving metallocenters that act as nucleophiles to form organometallic intermediates and catalyze C–C and C–S bond formations.

Background: Lipoxygenases (LOX) catalyze the oxygenation of polyunsaturated fatty acids but generate distinct products from a common substrate. Results: We report the first structure of a LOX-substrate complex. Conclusion:The structure provides a context for understanding product specificity in enzymes that metabolize arachidonic acid. Significance: With roles in the production of potent lipid mediators, LOX are targets for drug design.