Amy C. Rosenzweig

Nitrosopumilus maritimus genome reveals unique mechanisms for nitrification and autotrophy in globally distributed marine crenarchaea

Ammonia-oxidizing archaea are ubiquitous in marine and terrestrial environments and now thought to be significant contributors to carbon and nitrogen cycling. The isolation of Candidatus “Nitrosopumilus maritimus” strain SCM1 provided the opportunity for linking its chemolithotrophic physiology with a genomic inventory of the globally distributed archaea. Here we report the 1,645,259-bp closed genome of strain SCM1, revealing highly copper-dependent systems for ammonia oxidation and electron transport that are distinctly different from known ammonia-oxidizing bacteria. Consistent with in situ isotopic studies of marine archaea, the genome sequence indicates N. maritimus grows autotrophically using a variant of the 3-hydroxypropionate/4-hydroxybutryrate pathway for carbon assimilation, while maintaining limited capacity for assimilation of organic carbon. This unique instance of archaeal biosynthesis of the osmoprotectant ectoine and an unprecedented enrichment of multicopper oxidases, thioredoxin-like proteins, and transcriptional regulators points to an organism responsive to environmental cues and adapted to handling reactive copper and nitrogen species that likely derive from its distinctive biochemistry. The conservation of N. maritimus gene content and organization within marine metagenomes indicates that the unique physiology of these specialized oligophiles may play a significant role in the biogeochemical cycles of carbon and nitrogen.

Crystal structure of a membrane-bound metalloenzyme that catalyses the biological oxidation of methane.

Particulate methane monooxygenase (pMMO) is an integral membrane metalloenzyme that catalyses the conversion of methane to methanol. Knowledge of how pMMO performs this extremely challenging chemistry may have an impact on the use of methane as an alternative energy source by facilitating the development of new synthetic catalysts. We have determined the structure of pMMO from the methanotroph Methylococcus capsulatus (Bath) to a resolution of 2.8 Å. The enzyme is a trimer with an α3β3γ3 polypeptide arrangement. Two metal centres, modelled as mononuclear copper and dinuclear copper, are located in soluble regions of each pmoB subunit, which resembles cytochrome c oxidase subunit II. A third metal centre, occupied by zinc in the crystal, is located within the membrane. The structure provides new insight into the molecular details of biological methane oxidation.

Oxidation of methane by a biological dicopper centre

Vast world reserves of methane gas are underutilized as a feedstock for the production of liquid fuels and chemicals owing to the lack of economical and sustainable strategies for the selective oxidation of methane to methanol. Current processes to activate the strong C–H bond (104 kcal mol-1) in methane require high temperatures, are costly and inefficient, and produce waste. In nature, methanotrophic bacteria perform this reaction under ambient conditions using metalloenzymes called methane monooxygenases (MMOs). MMOs thus provide the optimal model for an efficient, environmentally sound catalyst. There are two types of MMO. Soluble MMO (sMMO) is expressed by several strains of methanotroph under copper-limited conditions and oxidizes methane with a well-characterized catalytic di-iron centre. Particulate MMO (pMMO) is an integral membrane metalloenzyme produced by all methanotrophs and is composed of three subunits, pmoA, pmoB and pmoC, arranged in a trimeric α3β3γ3 complex. Despite 20 years of research and the availability of two crystal structures, the metal composition and location of the pMMO metal active site are not known. Here we show that pMMO activity is dependent on copper, not iron, and that the copper active site is located in the soluble domains of the pmoB subunit rather than within the membrane. Recombinant soluble fragments of pmoB (spmoB) bind copper and have propylene and methane oxidation activities. Disruption of each copper centre in spmoB by mutagenesis indicates that the active site is a dicopper centre. These findings help resolve the pMMO controversy and provide a promising new approach to developing environmentally friendly C–H oxidation catalysts.

Structural basis for copper transfer by the metallochaperone for the Menkes/Wilson disease proteins.

The Hah1 metallochaperone protein is implicated in copper delivery to the Menkes and Wilson disease proteins. Hah1 and the N-termini of its target proteins belong to a family of metal binding domains characterized by a conserved MT/HCXXC sequence motif. The crystal structure of Hah1 has been determined in the presence of Cu(I), Hg(II), and Cd(II). The 1.8 Å resolution structure of CuHah1 reveals a copper ion coordinated by Cys residues from two adjacent Hah1 molecules. The CuHah1 crystal structure is the first of a copper chaperone bound to copper and provides structural support for direct metal ion exchange between conserved MT/HCXXC motifs in two domains. The structures of HgHah1 and CdHah1, determined to 1.75 Å resolution, also reveal metal ion coordination by two MT/HCXXC motifs. An extended hydrogen bonding network, unique to the complex of two Hah1 molecules, stabilizes the metal binding sites and suggests specific roles for several conserved residues. Taken together, the structures provide models for intermediates in metal ion transfer and suggest a detailed molecular mechanism for protein recognition and metal ion exchange between MT/HCXXC containing domains.

Geometry of the soluble methane monooxygenase catalytic diiron center in two oxidation states.

BACKGROUND The hydroxylase component of soluble methane monooxygenase (sMMO) contains a dinuclear iron center responsible for the oxidation of methane to methanol. As isolated, the center is in the oxidized, diiron(III) state. The 2.2 A resolution X-ray structure of the oxidized hydroxylase, Hox, from Methylococcus capsulatus (Bath) was previously determined at 4 degrees C. In this structure the two iron atoms are bridged by a glutamate, a hydroxide ion, and an acetate ion, and additionally coordinated to two His residues, three Glu residues, and a water molecule. RESULTS The 1.7 A resolution crystal structures of the sMMO hydroxylase from Methylococcus capsulatus (Bath) in both its oxidized diiron(III), Hox, and dithionite-treated, reduced diiron(II), Hred, oxidation states were determined at -160 degrees C. The structure of the diiron center in Hox differs from that previously reported at 2.2 A resolution and 4 degrees C. Although the hydroxide bridge is retained, the bidentate, bridging ligand assigned as acetate is replaced by a weakly coordinating monoatomic water bridge. In the resulting four-membered Fe(OH)Fe(OH2) ring, the Fe ... Fe distance is shortened from 3.4 A to 3.1 A. In protomer A of Hred, the hydroxide bridge is displaced by an oxygen atom of Glu243, which undergoes a carboxylate shift from its terminal monodentate binding mode in Hox to a mode in which the carboxylate is both monoatomic bridging and bidentate chelating. We therefore conclude that the center has been reduced to the diiron(II) oxidation state. Both iron atoms are coordinated to five ligands and weakly to a sixth water molecule in the resulting structure. The diiron center in protomer B of Hred has the same composition as those in Hox. In both the oxidized and reduced structures, the diiron core is connected through hydrogen bonds involving exogenous species to Thr213 in the active site cavity. CONCLUSIONS The diiron center in Hox can change its exogenous ligand coordination and geometry, a property that could be important in the catalytic cycle of sMMO. In Hred, a carboxylate shift occurs, extruding hydroxide ion and opening coordination sites for reaction with O2 to form the diiron(III) peroxo intermediate, Hperoxo. Residue Thr213 may function in catalysis.

Structural biology of copper trafficking.

1.1. Background The use of copper in biological systems coincides with the advent of an oxygen atmosphere about 1.7 billion years ago. The presence of O2 both allowed the oxidation of insoluble Cu(I) to the more soluble and bioavailable Cu(II) and led to the requirement for a redox active metal with potentials in the 0-800 mV range. Not only did copper meet this need, but the oxidation of Fe(II) to the insoluble Fe(III) form rendered the use of iron more energetically expensive.1-5 As a result, copper plays a key role in many proteins that react with O2. Generally, O2-reactive centers are mononuclear (type 2), dinuclear (type 3), or trinuclear (type 2 and type 3). Well studied mononuclear copper enzymes include the monooxygenases dopamine-β-hydroxylase and peptidylglycine α-hydroxylating monooxygenase as well as oxidases that also contain organic cofactors, such as amine, galactose, and lysyl oxidases.6 Dinuclear copper proteins include the O2 carrier hemocyanin and enzymes like tyrosinase and catechol oxidase.7 Copper also plays a key role in numerous electron transfer proteins. Mononuclear type 1 (blue copper) centers are found in proteins such as plastocyanin and azurin.8 The multicopper oxidases like laccase, ascorbate oxidase, and ceruloplasmin contain both a catalytic trinuclear type 2/type 3 site and an electron transfer type 1 site.9,10 The classification of copper centers into types is derived from optical and electron paramagnetic resonance (EPR) spectroscopic properties, and there are some notable exceptions, including the cysteine-bridged dinuclear CuA electron transfer site in cytochrome c oxidase11 and nitrous oxide reductase, the tetranuclear catalytic CuZ center in nitrous oxide reductase,12 and the proposed catalytic copper center in particulate methane monooxygenase.13-15 The same redox properties that render copper useful in all these metalloproteins can lead to oxidative damage in cells. Reaction of Cu(I) with hydrogen peroxide and re-reduction of Cu(II) by superoxide via Fenton and Haber-Weiss chemistry yields hydroxyl radicals that can damage proteins, lipids, and nucleic acids.16 Thus, intracellular copper concentrations must be controlled such that copper ions are provided to essential enzymes, but do not accumulate to deleterious levels. In humans, deficiencies in copper metabolism are linked to diseases such as Menkes syndrome, Wilson disease, prion diseases, and Alzheimer’s disease.17 Several classes of proteins, including membrane transporters,18-20 metallochaperones,21,22 and metalloregulatory proteins,23,24 are implicated in copper homeostasis. These proteins have two functions. First, they ensure that copper is provided to the correct proteins and cellular compartments for necessary activities. Second, these proteins detoxify excess copper. Just as copper-containing proteins and enzymes are found in all kingdoms of life, members of these groups of homeostatic proteins are also widespread,5 and have been structurally and biochemically characterized from eukaryotes and prokaryotes.

Heterodimeric structure of superoxide dismutase in complex with its metallochaperone.

The copper chaperone for superoxide dismutase (CCS) activates the eukaryotic antioxidant enzyme copper, zinc superoxide dismutase (SOD1). The 2.9 Å resolution structure of yeast SOD1 complexed with yeast CCS (yCCS) reveals that SOD1 interacts with its metallochaperone to form a complex comprising one monomer of each protein. The heterodimer interface is remarkably similar to the SOD1 and yCCS homodimer interfaces. Striking conformational rearrangements are observed in both the chaperone and target enzyme upon complex formation, and the functionally essential C-terminal domain of yCCS is well positioned to play a key role in the metal ion transfer mechanism. This domain is linked to SOD1 by an intermolecular disulfide bond that may facilitate or regulate copper delivery.

Crystal structure of the Atx1 metallochaperone protein at 1.02 Å resolution

BACKGROUND Metallochaperone proteins function in the trafficking and delivery of essential, yet potentially toxic, metal ions to distinct locations and particular proteins in eukaryotic cells. The Atx1 protein shuttles copper to the transport ATPase Ccc2 in yeast cells. Molecular mechanisms for copper delivery by Atx1 and similar human chaperones have been proposed, but detailed structural characterization is necessary to elucidate how Atx1 binds metal ions and how it might interact with Ccc2 to facilitate metal ion transfer. RESULTS The 1.02 A resolution X-ray structure of the Hg(II) form of Atx1 (HgAtx1) reveals the overall secondary structure, the location of the metal-binding site, the detailed coordination geometry for Hg(II), and specific amino acid residues that may be important in interactions with Ccc2. Metal ion transfer experiments establish that HgAtx1 is a functional model for the Cu(I) form of Atx1 (CuAtx1). The metal-binding loop is flexible, changing conformation to form a disulfide bond in the oxidized apo form, the structure of which has been solved to 1.20 A resolution. CONCLUSIONS The Atx1 structure represents the first structure of a metallochaperone protein, and is one of the largest unknown structures solved by direct methods. The structural features of the metal-binding site support the proposed Atx1 mechanism in which facile metal ion transfer occurs between metal-binding sites of the diffusible copper-donor and membrane-tethered copper-acceptor proteins. The Atx1 structural motif represents a prototypical metal ion trafficking unit that is likely to be employed in a variety of organisms for different metal ions.

Metallochaperones: Bind and Deliver

Metallochaperones deliver metal ions directly to target proteins via specific protein-protein interactions. Recent research has led to a molecular picture of how some metallochaperones bind metal ions, recognize their partner proteins, and accomplish metal ion transfer.

Crystal structure of the copper chaperone for superoxide dismutase

Cellular systems for handling transition metal ions have been identified, but little is known about the structure and function of the specific trafficking proteins. The 1.8 Å resolution structure of the yeast copper chaperone for superoxide dismutase (yCCS) reveals a protein composed of two domains. The N-terminal domain is very similar to the metallochaperone protein Atx1 and is likely to play a role in copper delivery and/or uptake. The second domain resembles the physiological target of yCCS, superoxide dismutase I (SOD1), in overall fold, but lacks all of the structural elements involved in catalysis. In the crystal, two SOD1-like domains interact to form a dimer. The subunit interface is remarkably similar to that in SOD1, suggesting a structural basis for target recognition by this metallochaperone.