Methanotrophic bacteria are capable of using methane as their sole source of carbon and energy. The first step in methane metabolism, the oxidation of methane to methanol, is catalyzed by a fascinating enzyme system called methane monooxygenase (MMO). The selective oxidation of the very stable C-H bond in methane under ambient conditions is a remarkable feat that has not yet been repeated by synthetic catalysts and has attracted considerable scientific and commercial interest. The best studied MMO is a complex enzyme system that consists of three soluble protein components, all of which are required for efficient catalysis. Dioxygen activation and subsequent methane hydroxylation are catalyzed by a hydroxylase enzyme that contains a non-heme diiron site. A reductase protein accepts electrons from NADH and transfers them to the hydroxylase where they are used for the reductive activation of O(2). The third protein component couples electron and dioxygen consumption with methane oxidation. In this review we examine different aspects of catalysis by the MMO proteins, including the mechanisms of dioxygen activation at the diiron site and substrate hydroxylation by the activated oxygen species. We also discuss the role of complex formation between the different protein components in regulating various aspects of catalysis.

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The four-component toluene/o-xylene monooxygenase (ToMO) from Pseudomonas stutzeri OX1 is capable of oxidizing arenes, alkenes, and haloalkanes at a carboxylate-bridged diiron center similar to that of soluble methane monooxygenase (sMMO). The remarkable variety of substrates accommodated by ToMO invites applications ranging from bioremediation to the regioand enantiospecific oxidation of hydrocarbons on an industrial scale. We report here the crystal structures of the ToMO hydroxylase (ToMOH), azido ToMOH, and ToMOH containing the product analogue 4-bromophenol to 2.3 Å or greater resolution. The catalytic diiron(III) core resembles that of the sMMO hydroxylase, but aspects of the ␣ 2 ␤ 2 ␥ 2 tertiary structure are notably different. Of particular interest is a 6 -10 Å-wide channel of ϳ35 Å in length extending from the active site to the protein surface. The presence of three bromophenol molecules in this space confirms this route as a pathway for substrate entrance and product egress. An analysis of the ToMOH active site cavity offers insights into the different substrate specificities of multicomponent monooxygenases and explains the behavior of mutant forms of homologous enzymes described in the literature.Bacterial multicomponent monooxygenases (BMMs) 1 comprise a family of carboxylate-bridged non-heme diiron enzymes capable of oxidizing a broad range of hydrocarbons including C 1 -C 8 alkanes, alkenes, and aromatics (1, 2). Four characterized subclasses of multicomponent monooxygenases have been defined (2, 3). These are soluble methane monooxygenases (sMMOs), four-component alkene/arene monooxygenases or toluene monooxygenases (TMOs), three-component phenol hydroxylases (PHs), and ␣␤ alkene monoxygenases (AMOs), of which all are believed to have evolved from a common ancestor. Bacteria containing multicomponent monooxygenases are capable of using specific hydrocarbon substrates as their primary source of carbon and energy (1, 2, 4). The remarkable range of substrate specificity exhibited by these enzymes endows these bacteria with the ability to bioremediate environmentally harmful substances such as trichloroethylene and petroleum spills (5, 6) and to regulate the global carbon cycle (4). BMMs can also perform regio-and stereospecific hydroxylations, making them useful for producing pure feedstocks for industrial synthesis (7). These enzyme systems, although highly homologous, have evolved different substrate specificities. Only soluble methane monooxygenase can activate the inert C-H bond of methane, which is one of the most difficult reactions to perform in nature (1), whereas the catalytic abilities of TMOs are limited to aromatics, alkenes, and some haloalkanes (2, 5).Substrate hydroxylation in BMMs occurs at a dioxygen-activated, carboxylate-bridged diiron center in the ␣-subunit of a ϳ220 -250 kDa hydroxylase component that is an (␣␤␥) 2 heterodimer or, in the case of one known AMO, an ␣␤ monomer (1-3, 8, 9). Sequence identity comparisons and spectroscopic studies suggest that the diiron centers of...

Bacterial multicomponent monooxygenases (BMMs) catalyze the O2-dependent hydroxylation of hydrocarbons at a carboxylate-bridged diiron center similar to those that occur in a variety of dimetallic oxygen-utilizing enzymes. BMMs have found numerous biodegradation and biocatalytic applications. Recent investigations have begun to reveal how BMMs perform their C-H bond activation chemistry and why these enzymes may be mechanistically different from other related diiron proteins. The structures of the BMM component proteins and of complexes between them provide insights into the tuning of the dinuclear iron center and the enzyme mechanism. Selected findings are compared and contrasted with the properties of other carboxylate-bridged diiron proteins, revealing common structural and functional themes.

Phenol hydroxylase (PH) belongs to a family of bacterial multicomponent monooxygenases (BMMs) with carboxylate-bridged diiron active sites. Included are toluene/o-xylene (ToMO) and soluble methane (sMMO) monooxygenase. PH hydroxylates aromatic compounds, but unlike sMMO, it cannot oxidize alkanes despite having a similar dinuclear iron active site. Important for activity is formation of a complex between the hydroxylase and a regulatory protein component. To address how structural features of BMM hydroxylases and their component complexes may facilitate the catalytic mechanism and choice of substrate, we determined X-ray structures of native and SeMet forms of the PH hydroxylase (PHH) in complex with its regulatory protein (PHM) to 2.3 Å resolution. PHM binds in a canyon on one side of the (αβγ) 2 PHH dimer, contacting α-subunit helices A, E, and F ∼12 Å above the diiron core. The structure of the dinuclear iron center in PHH resembles that of mixed-valent MMOH, suggesting an Fe(II)Fe(III) oxidation state. Helix E, which comprises part of the iron-coordinating four-helix bundle, has more π-helical character than analogous E helices in MMOH and ToMOH lacking a bound regulatory protein. Consequently, conserved active site Thr and Asn residues translocate to the protein surface, and an ∼6 Å pore opens through the four-helix bundle. Of likely functional significance is a specific hydrogen bond formed between this Asn residue and a conserved Ser side chain on PHM. The PHM protein covers a putative docking site on PHH for the PH reductase, which transfers electrons to the PHH diiron center prior to O 2 activation, † This research was supported by National Institute of General Medical Sciences Grant GM32134 (S.J.L.) and the Italian Ministry of SUPPORTING INFORMATION AVAILABLE BMM hydroxylase and regulatory protein structures and their electrostatic surfaces (Figures S1 and S2), packing of the β-subunit Nterminus and a γ-subunit from an adjacent molecule in the PHH canyon ( Figure S3), comparison of known regulatory protein structures ( Figure S4), sequence alignments of the different regulatory proteins ( Figure S5), models of the PHH diiron center in electron density maps ( Figure S6), comparison of the hydroxylase zinc-binding sequences ( Figure S7), and electron density maps around helix F ( Figure S8). This material is available free of charge via the Internet at http://pubs.acs.org. NIH Public Access Author ManuscriptBiochemistry. Author manuscript; available in PMC 2007 March 21. NIH-PA Author ManuscriptNIH-PA Author Manuscript NIH-PA Author Manuscriptsuggesting that the regulatory component may function to block undesired reduction of oxygenated intermediates during the catalytic cycle. A series of hydrophobic cavities through the PHH α-subunit, analogous to those in MMOH, may facilitate movement of the substrate to and/or product from the active site pocket. Comparisons between the ToMOH and PHH structures provide insights into their substrate regiospecificities.Bacterial multicomponent monooxygenases...

The soluble methane monooxygenase hydroxylase (MMOH) alpha-subunit contains a series of cavities that delineate the route of substrate entrance to and product egress from the buried carboxylate-bridged diiron center. The presence of discrete cavities is a major structural difference between MMOH, which can hydroxylate methane, and toluene/o-xylene monooxygenase hydroxylase (ToMOH), which cannot. To understand better the functions of the cavities and to investigate how an enzyme designed for methane hydroxylation can also accommodate larger substrates such as octane, methylcubane, and trans-1-methyl-2-phenylcyclopropane, MMOH crystals were soaked with an assortment of different alcohols and their X-ray structures were solved to 1.8-2.4 A resolution. The product analogues localize to cavities 1-3 and delineate a path of product exit and/or substrate entrance from the active site to the surface of the protein. The binding of the alcohols to a position bridging the two iron atoms in cavity 1 extends and validates previous crystallographic, spectroscopic, and computational work indicating this site to be where substrates are hydroxylated and products form. The presence of these alcohols induces perturbations in the amino acid side-chain gates linking pairs of cavities, allowing for the formation of a channel similar to one observed in ToMOH. Upon binding of 6-bromohexan-1-ol, the pi helix formed by residues 202-211 in helix E of the alpha-subunit is extended through residue 216, changing the orientations of several amino acid residues in the active site cavity. This remarkable secondary structure rearrangement in the four-helix bundle has several mechanistic implications for substrate accommodation and the function of the effector protein, MMOB.

The P-type ATPases translocate cations across membranes using the energy provided by ATP hydrolysis. CopA from Archaeoglobus fulgidus is a hyperthermophilic ATPase responsible for the cellular export of Cu ؉ and is a member of the heavy metal P 1B -type ATPase (3,4). Widely distributed in nature, the P 1B -type ATPases confer heavy metal tolerance to microorganisms (5, 6) and are essential for the absorption, distribution, and bioaccumulation of metal micronutrients by multicellular eukaryotes (7,8). The importance of these enzymes for metal homeostasis is underscored by the two Cu ϩ -ATPases present in humans, ATP7A (MNK) and ATP7B (WND) (7, 9). Mutations in the genes encoding the ATP7A and ATP7B proteins lead to Menkes and Wilson diseases, respectively. Menkes disease is characterized by impaired transport of dietary copper across the small intestine, resulting in a copper deficiency in peripheral tissues. Improper function of WND leads to toxic copper accumulation in the liver because of reduced biliary excretion (10, 11). Although all P-type ATPases share essential structural elements, the subgroups differ in the number of predicted transmembrane helices and the arrangement of these segments with respect to the cytoplasmic ATP binding domain (ATPBD). 4 In addition, the soluble regions vary in length and composition (4, 5, 12). The P 1B -type ATPases comprise eight transmembrane helices, a large ATPBD, an actuator domain (A-domain), and N-or C-terminal soluble metal binding domains (MBDs). Binding sites responsible for metal coordination during transport are located within the membrane and are believed to involve a CPX sequence motif as well as other key residues that confer metal ion selectivity (3, 13). The N-terminal MBDs (N-MBDs), ranging from one to six depending on the organism, receive copper ions from metallochaperones (14), participate in ATPase regulation (15), and facilitate intracellular relocalization (16). In the absence of copper, the six WND N-MBDs interact with the ATPBD (17), suggesting distinct mechanisms of regulation by substrates in P 1B -type ATPases.The P 1B -type ATPase transport mechanism follows the classical PostAlbers E1/E2 model (7,18,19). The central element of this catalytic cycle is the coupling of metal transport to enzyme phosphorylation by ATP. Phosphorylation of the aspartic acid residue from the signature sequence DKTGT is the unifying characteristic of all P-type ATPases (1, 2). The ATP binding, hydrolysis, and enzyme phosphorylation steps occur within the ATPBD, which consists of a phosphorylation domain (P-domain) and a nucleotide binding domain (N-domain). The ATPBD from WND (17), as well as those from P 2 and P 1A -ATPases (20 -22), have been isolated in soluble form. These cytoplasmic fragments are able to bind nucleotides and hydrolyze ATP, albeit at a low rate, and thus contain the key components needed for energy dependent ion transport by these ATPases.With the exception of solution structures of N-MBDs (23, 24), structural information for P 1B -type ATPases is...