Methane oxidation is extremely difficult chemistry to perform in the laboratory. The CÀH bond in CH 4 has the highest bond energy (104 kcal mol À1 ) amongst organic substrates. In nature, the controlled oxidation of organic substrates is mediated by an important class of enzymes known as monooxygenases and dioxygenases, [1] and the methane monooxygenases are unique in their capability to mediate the facile conversion of methane to methanol. [2,3] With a turnover frequency approaching 1 s À1 , the particulate methane monooxygenase (pMMO) is the most efficient methane oxidizer discovered to date. Given the current interest in developing a laboratory catalyst suitable for the conversion of methane to methanol on an industrial scale, there is strong impetus to understand how pMMO works and to develop functional biomimetics of this enzyme. pMMO is a complex membrane protein consisting of three subunits (PmoA, PmoB, and PmoC) and many copper cofactors. [3] Inspired by the proposal that the catalytic site might be a tricopper cluster, we have recently developed a series of tricopper complexes that are capable of supporting facile catalytic oxidation of hydrocarbons. [4,5] We show herein that these model tricopper complexes can mediate efficient catalytic oxidation of methane to methanol as well.The oxidation of CH 4 mediated by the tricopper complex [Cu I Cu I Cu I (7-N-Etppz)] 1+ in acetonitrile (ACN), where 7-N-Etppz corresponds to the ligand 3,3'-(1,4-diazepane-1,4diyl)bis[1-(4-ethylpiperazine-1-yl)propan-2-ol], is summarized in Figure 1 A. A single turnover (turnover number; TON = 0.92) is obtained when this Cu I Cu I Cu I complex is activated by excess dioxygen in the presence of excess CH 4 (Figure 1 B). The reaction is complete within ten minutes, clearly indicating that the oxidation is very rapid. In accordance with the single turnover, the kinetics of the overall process is pseudo first-order with respect to the concentration of the fully reduced tricopper complex with a rate constant k 1 = 0.065 min À1 (Figure 1 B, inset). If we assume that the kinetics is limited by the dioxygen activation of the Cu I Cu I Cu I cluster with the subsequent O-atom transfer to the substrate molecule being rapid, then k 1 = k 2 ·[O 2 ] 0 , and from the solubility of oxygen in ACN at 25 8C (8.1 mm), [6] we obtain the bimolecular rate constant k 2 of 1.33 10 À1 m À1 s À1 for the dioxygen activation of the Cu I Cu I Cu I cluster. This second-order rate constant is similar to values that we have previously determined for the dioxygen activation of other model tricopper clusters at room temperature. [7,8] The process can be made catalytic by adding the appropriate amounts of H 2 O 2 to regenerate the spent catalyst after O-atom transfer from the activated tricopper complex to CH 4 . This multiple-turnover reaction is depicted in Figure 1 C. In these experiments, the [Cu I Cu I Cu I (7-N-Etppz)] 1+ catalyst is activated by O 2 as in the single-turnover experiment described earlier, but the spent catalyst is regenerated by twoelectron ...

In order to obtain particulate methane monooxygenase (pMMO)-enriched membranes from Methylococcus capsulatus (Bath) with high activity and in high yields, we devised a method to process cell growth in a fermentor adapted with a hollow-fiber bioreactor that allows easy control and quantitative adjustment of the copper ion concentration in NMS medium over the time course of cell culture. This technical improvement in the method for culturing bacterial cells allowed us to study the effects of copper ion concentration in the growth medium on the copper content in the membranes, as well as the specific activity of the enzyme. The optimal copper concentration in the growth medium was found to be 30 to 35 M. Under these conditions, the pMMO is highly expressed, accounting for 80% of the total cytoplasmic membrane proteins and having a specific activity as high as 88.9 nmol of propylene oxide/min/mg of protein with NADH as the reductant. The copper stoichiometry is ϳ13 atoms per pMMO molecule. Analysis of other metal contents provided no evidence of zinc, and only traces of iron were present in the pMMO-enriched membranes. Further purification by membrane solubilization in dodecyl ␤-D-maltoside followed by fractionation of the protein-detergent complexes according to molecular size by gel filtration chromatography resulted in a good yield of the pMMO-detergent complex and a high level of homogeneity. The pMMO-detergent complex isolated in this way had a molecular mass of 220 kDa and consisted of an ␣␤␥ protein monomer encapsulated in a micelle consisting of ca. 240 detergent molecules. The enzyme is a copper protein containing 13.6 mol of copper/mol of pMMO and essentially no iron (ratio of copper to iron, 80:1). Both the detergent-solubilized membranes and the purified pMMO-detergent complex exhibited reasonable, if not excellent, specific activity. Finally, our ability to control the level of expression of the pMMO allowed us to clarify the sensitivity of the enzyme to NADH and duroquinol, the two common reductants used to assay the enzyme.

The development of a heterogeneous catalyst capable for efficient selective conversion of methane into methanol with multiple turnovers under ambient conditions is reported here.

Copper ions switch the oxidation of methane by soluble methane monooxygenase to particulate methane monooxygenase in Methylococcus capsulatus (Bath). Toward understanding the change in cellular metabolism related to this transcriptional and metabolic switch, we have undertaken genomic sequencing and quantitative comparative analysis of the proteome in M. capsulatus (Bath) grown under different copper-to-biomass ratios by cleavable isotope-coded affinity tag technology. Of the 682 proteins identified, the expressions of 60 proteins were stimulated by at least 2-fold by copper ions; 68 proteins were down-regulated by 2-fold or more. The 60 proteins overexpressed included the methane and carbohydrate metabolic enzymes, while the 68 proteins suppressed were mainly responsible for cellular signaling processes, indicating a role of copper ions in the expression of the genes associated with the metabolism of the organism downstream of methane oxidation. The study has also provided a complete map of the C 1 metabolism pathways in this methanotroph and clarified the interrelationships between them.Methanotrophs are a unique group of Gram-negative bacteria that grow aerobically on methane and utilize methane as the sole source of carbon and energy. There has been considerable interest in methanotrophs over the past 30 years, since they can be used to produce single-cell bulk chemicals such as propylene oxide. The ability of these bacteria to co-oxidize a wide range of alkanes, alkenes, and substituted aliphatic compounds has been exploited in bioremediation processes, for example, in the degradation of key pollutants such as trichloroethylene in soil and groundwater. Methanotrophs also play an important role in the global methane cycle (1, 2).Methanotrophs are classified into three distinct types. Type I methanotrophs show disk-like intracellular membranes and use the RuMP (ribulose 5-phosphate) pathway for carbon assimilation. In contrast, Type II methanotrophs possess intracellular membranes in the cell periphery and use the serine pathway to degrade the formaldehyde produced. Methylococcus capsulatus (Bath) is classified as a Type X methanotroph (1), since it shows the physiological properties of both Type I and II methanotrophs, but it develops Type I intracytoplasmic membranes.Two methane monooxygenases (MMO) 1 catalyze the methane oxidation process in methanotrophs, converting methane to methanol. All methanotrophs express a membrane-bound copper-containing particulate MMO (pMMO) (1, 3), while Type X and a few Type II methanotrophic bacteria are capable of producing a second, soluble form (soluble methane monooxygenase, sMMO). Copper ions are known to switch the methane oxidation from sMMO to pMMO. At low copper-to-biomass ratios, the cytoplasmic sMMO is the dominant MMO. When the cells are grown at high copper-to-biomass ratios, pMMO is expressed and produced instead in the plasma membrane.Because of the unique role of copper ions in regulating the switch between sMMO and pMMO, we have applied cICAT (cleavable isot...

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Statherin is an active inhibitor of calcium phosphate precipitation in the oral cavity. For many studies of the interaction between statherin and hydroxyapatite (HAp), the samples are prepared by a direct mixing of statherin or its fragment with well-crystalline HAp crystals. In this work, the HAp sample is precipitated in the presence of peptide fragment derived from the N-terminal 15 amino acids of statherin (SN-15). The in situ prepared HAp crystallites are nanosized, leading to a significant increase of the peptide amount adsorbed on the HAp surface. The enhancement in NMR sensitivity allows, for the first time, the measurement of a two-dimensional 13C-13C correlation spectrum for a 13C uniformly labeled peptide sample adsorbed on mineral surface. The measurement time is about 18.5 h at a field strength of 7.05 T. Preliminary results suggest that there may exist two different mechanisms for the interaction between SN-15 and HAp. In addition to the one which will cause a conformational change near the N-terminal, SN-15 may also be absorbed on the HAp surface by simple electrostatic interaction, without any significant conformational changes of the peptides.

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Highlights  Subunit B (PmoB) of particulate methane monooxygenase (pMMO) is expressed in E. coli.  PmoB and its variants/mutants are expressed in the membranes as Cu I proteins.  The PmoB of pMMO contains a Cu I sponge with high reduction potentials for the Cu sites.  The PmoB proteins show evidence of a dinuclear copper site.  The PmoB-enriched E. coli membranes produce H 2 O 2 .