Improvements in purification of membrane-associated methane monooxygenase (pMMO) have resulted in preparations of pMMO with activities more representative of physiological rates: i.e., >130 nmol ⅐ min ؊1 ⅐ mg of protein ؊1 . Altered culture and assay conditions, optimization of the detergent/protein ratio, and simplification of the purification procedure were responsible for the higher-activity preparations. Changes in the culture conditions focused on the rate of copper addition. To document the physiological events that occur during copper addition, cultures were initiated in medium with cells expressing soluble methane monooxygenase (sMMO) and then monitored for morphological changes, copper acquisition, fatty acid concentration, and pMMO and sMMO expression as the amended copper concentration was increased from 0 (approximately 0.3 M) to 95 M. The results demonstrate that copper not only regulates the metabolic switch between the two methane monooxygenases but also regulates the level of expression of the pMMO and the development of internal membranes. With respect to stabilization of cell-free pMMO activity, the highest cell-free pMMO activity was observed when copper addition exceeded maximal pMMO expression. Optimization of detergent/ protein ratios and simplification of the purification procedure also contributed to the higher activity levels in purified pMMO preparations. Finally, the addition of the type 2 NADH:quinone oxidoreductase complex (NADH dehydrogenase [NDH]) from M. capsulatus Bath, along with NADH and duroquinol, to enzyme assays increased the activity of purified preparations. The NDH and NADH were added to maintain a high duroquinol/duroquinone ratio.Methanotrophs are a group of gram-negative bacteria that utilize methane or methanol as the sole source of carbon and energy (1,20). The initial oxidation of methane to methanol is catalyzed by methane monooxygenase (MMO). In some methanotrophs, two different MMOs can be expressed, depending on the copper concentration during growth (11, 37, 39): a soluble cytoplasmic MMO (sMMO) and a membrane-associated, or particulate, MMO (pMMO). In cells cultured under low copper/biomass ratios (Յ0.9 nmol of Cu/mg of cell protein), the sMMO is expressed (20, 28). Cells cultured under higher copper/biomass ratios express pMMO, and there is no detectable sMMO expression (35,43). While sMMO is a wellcharacterized enzyme that consists of a hydroxylase component composed of three polypeptides and a hydroxo-bridged binuclear iron cluster-an NADH-dependent reductase component composed of one polypeptide containing both FAD and [Fe 2 S 2 ] cofactors and a regulatory polypeptide (18,26,27,31,47)-information on the molecular properties of pMMO is limited due to the instability of pMMO in cell-free fractions.Purification of the pMMO has been reported from Methylococcus capsulatus Bath (2,25,33, 52) and M. trichosporium

To examine the potential role of methanobactin (mb) as the extracellular component of a copper acquisition system in Methylosinus trichosporium OB3b, the metal binding properties of mb were examined. Spectral (UV-visible, fluorescence, and circular dichroism), kinetic, and thermodynamic data suggested copper coordination changes at different Cu(II):mb ratios. Mb appeared to initially bind Cu(II) as a homodimer with a comparatively high copper affinity at Cu(II):mb ratios below 0.2, with a binding constant (K) greater than that of EDTA (log K = 18.8) and an approximate DeltaG degrees of -47 kcal/mol. At Cu(II):mb ratios between 0.2 and 0.45, the K dropped to (2.6 +/- 0.46) x 10(8) with a DeltaG degrees of -11.46 kcal/mol followed by another K of (1.40 +/- 0.21) x 10(6) and a DeltaG degrees of -8.38 kcal/mol at Cu(II):mb ratios of 0.45-0.85. The kinetic and spectral changes also suggested Cu(II) was initially coordinated to the 4-thiocarbonyl-5-hydroxy imidazolate (THI) and possibly Tyr, followed by reduction to Cu(I), and then coordination of Cu(I) to 4-hydroxy-5-thiocarbonyl imidazolate (HTI) resulting in the final coordination of Cu(I) by THI and HTI. The rate constant (k(obsI)) of binding of Cu(II) to THI exceeded that of the stopped flow apparatus that was used, i.e., >640 s(-)(1), whereas the coordination of copper to HTI showed a 6-8 ms lag time followed by a k(obsII) of 121 +/- 9 s(-)(1). Mb also solubilized and bound Cu(I) with a k(obsI) to THI of >640 s(-)(1), but with a slower rate constant to HTI (k(obsII) = 8.27 +/- 0.16 s(-)(1)), and appeared to initially bind Cu(I) as a monomer.

Methanobactin (mb) is a small copper-binding peptide produced by methanotrophic bacteria and is intimately involved in both their copper metabolism and their role in the global carbon cycle. The structure for methanobactin comprises seven amino acids plus two chromophoric residues that appear unique to methanobactin. In a previously published structure, both chromophoric residues contain a thiocarbonyl attached to a hydroxyimidazolate ring. In addition, one is attached to a pyrrolidine ring, while the other to an isopropyl ester. A published X-ray determined structure for methanobactin shows these two chromophoric groups forming an N2S2 binding site for a single Cu(I) ion with distorted tetrahedral geometry. In this report we show that NMR, mass spectrometry, and chemical data, reveal a chemical structure that is significantly different than the previously published one. Specifically, the 1H and 13C NMR assignments are inconsistent with an N-terminal isopropyl ester and point instead to a 3-methylbutanoyl group. Our data also indicate that oxazolone rings instead of hydroxyimidazolate rings form the core of the two chromophoric residues. Because these rings are directly involved in the binding of Cu(I) and other metals by methanobactin, and are likely involved in the many chemical activities displayed by methanobactin, their correct identity is central to developing an accurate and detailed understanding of methanobactin’s many chemical and biological roles. For example, the oxazolone rings make methanobactin structurally more similar to other bacterially produced bactins and siderophores and suggest pathways for its biosynthesis.

Methane monooxygenase (MMO) catalyzes the energy dependent oxidation of methane to methanol in methanotrophic bacteria. 1, 2 In these organisms two different methane monooxygenases have been identified, namely a membrane-associated or particulate MMO (pMMO) and a cytoplasmic or soluble MMO (sMMO). In methanotrophs that express both forms of the enzyme, the copper concentration during growth dictates which MMO is expressed. 2-5 In cells cultured under a low copper/biomass ratio, the sMMO is predominately expressed, whereas cells cultured at higher copper/biomass ratios exclusively express the pMMO (sMMO is not transcribed). 6-8 The sMMO is a well characterized three-component enzyme consisting of a hydroxylase, a reductase and a regulatory protein. 9-12 Spectroscopic and X-ray crystallographic studies have established that the hydroxylase contains an oxygen bridged diiron cluster. 13-16 Here we provide evidence that pMMO contains a diiron cluster as well.Owing to the low specific activity and instability of most pMMO preparations, 6, 17-20 comparatively little is known about the molecular properties of this enzyme. As isolated, pMMO is composed of three polypeptides with molecular masses of 45,000, 26,000, and 23,000 Da with a subunit structure of (αβγ) 3 . 6, 17, 18, 20-22 Most researchers agree that each αβγ contains 2 -3 Cu atoms 2, 6, 17-20, 23 although one group has suggested that 15 Cu atoms are arranged into catalytic and electron transfer trinuclear copper clusters. 22, 24, 25 The 2.8 Å resolution crystal structure of pMMO revealed that each αβγ contained a dicopper site, a mononuclear copper site, and a third site occupied by zinc. 21, 23 However, the preparation used for growing the crystal was inactive and did not contain zinc (which was added to the crystallization buffer). 21, 23The involvement of non-heme iron in methane oxidation by the pMMO has been proposed by some laboratories 6, 17, 26-29 and disputed by others. 22, 24, 30 In our laboratory at Iowa State University we have observed that preparations of pMMO showing highest specific activity contain 1-2 iron atoms. 6 We therefore decided to employ Mössbauer spectroscopy to characterize the iron components. This technique is particularly well suited to explore iron environments that are EPR-silent and optically uninformative in the visible region, as is the Author E-mail : emunck@cmu.edu. Table 1 lists analytical and activity data of our purified pMMO sample and of whole cells grown at high copper (80μM) and iron (40μM); the entries are discussed in the Supporting Information. Figure 1 shows 4.2 K Mössbauer spectra of purified pMMO. The central portion of the 45 mT spectrum ( Figure 1A) exhibits two overlapping doublets with ΔE Q (1) = 1.05 mm/ s, δ(1) = 0.50 mm/s (≈20% of total Fe) and ΔE Q (2) = 2.65 mm/s, δ(1) = 1.25 mm/s (≈18% of total Fe); the δ value of doublet 2 is characteristic of a high-spin Fe 2+ with octahedral N/O coordination. The majority of the iron in the spectrum of Figure 1A, perhaps up to 60% of total Fe, belongs to a heter...

Improvements in the purification of methanobactin (mb) from either Methylosinus trichosporium OB3bT or Methylococcus capsulatus Bath resulted in preparations that stimulated methane-oxidation activity in both whole-cell and cell-free fractions of Methylococcus capsulatus Bath expressing the membrane-associated methane monooxygenase (pMMO). By using washed membrane factions with pMMO activities in the 290 nmol propylene oxidized min "1 (mg protein) "1 range, activities approaching 400 nmol propylene oxidized min "1 (mg protein) "1 were commonly observed following addition of copper-containing mb (Cu-mb), which represented 50-75 % of the total whole-cell activity.The stimulation of methane-oxidation activity by Cu-mb was similar to or greater than that observed with equimolar concentrations of Cu(II), without the inhibitory effects observed with high copper concentrations. Stimulation of pMMO activity was not observed with copper-free mb, nor was it observed when the copper-to-mb ratio was <0?5 Cu atoms per mb. The electron paramagnetic resonance (EPR) spectra of mb differed depending on the copper-to-mb ratio. At copper-to-mb ratios of <0?4 Cu(II) per mb, Cu(II) addition to mb showed an initial coordination by both sulfur and nitrogen, followed by reduction to Cu(I) in <2 min. At Cu(II)-to-mb ratios between 0?4 and 0?9 Cu(II) per mb, the intensity of the Cu(II) signal in EPR spectra was more representative of the Cu(II) added and indicated more nitrogen coordination. The EPR spectral properties of mb and pMMO were also examined in the washed membrane fraction following the addition of Cu(II), mb and Cu-mb in the presence or absence of reductants (NADH or duroquinol) and substrates (CH 4 and/or O 2 ). The results indicated that Cu-mb increased electron flow to the pMMO, increased the free radical formed following the addition of O 2 and decreased the residual free radical following the addition of O 2 plus CH 4 . The increase in pMMO activity and EPR spectral changes to the pMMO following Cu-mb addition represent the first positive evidence of interactions between the pMMO and Cu-mb.

Planar perovskite solar cells using low‐temperature atomic layer deposition (ALD) of the SnO2 electron transporting layer (ETL), with excellent electron extraction and hole‐blocking ability, offer significant advantages compared with high‐temperature deposition methods. The optical, chemical, and electrical properties of the ALD SnO2 layer and its influence on the device performance are investigated. It is found that surface passivation of SnO2 is essential to reduce charge recombination at the perovskite and ETL interface and show that the fabricated planar perovskite solar cells exhibit high reproducibility, stability, and power conversion efficiency of 20%.

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