Steve S.-F. Yu

Production of High-Quality Particulate Methane Monooxygenase in High Yields from Methylococcus capsulatus (Bath) with a Hollow-Fiber Membrane Bioreactor

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 micro 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 approximately 13 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 beta-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 alphabetagamma 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.

Efficient Oxidation of Methane to Methanol by Dioxygen Mediated by Tricopper Clusters

Methane oxidation is extremely difficult chemistry to perform in the laboratory. The C H bond in CH4 has the highest bond energy (104 kcalmol ) amongst organic substrates. In nature, the controlled oxidation of organic substrates is mediated by an important class of enzymes known as monooxygenases and dioxygenases, and the methane monooxygenases are unique in their capability to mediate the facile conversion of methane to methanol. With a turnover frequency approaching 1 s , 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. 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. We show herein that these model tricopper complexes can mediate efficient catalytic oxidation of methane to methanol as well. The oxidation of CH4 mediated by the tricopper complex [CuCuCu(7-N-Etppz)] in acetonitrile (ACN), where 7-NEtppz corresponds to the ligand 3,3’-(1,4-diazepane-1,4diyl)bis[1-(4-ethylpiperazine-1-yl)propan-2-ol], is summarized in Figure 1A. A single turnover (turnover number; TON= 0.92) is obtained when this CuCuCu complex is activated by excess dioxygen in the presence of excess CH4 (Figure 1B). 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 k1= 0.065 min 1 (Figure 1B, inset). If we assume that the kinetics is limited by the dioxygen activation of the CuCuCu cluster with the subsequent O-atom transfer to the substrate molecule being rapid, then k1=k2·[O2]0, and from the solubility of oxygen in ACN at 25 8C (8.1 mm), we obtain the bimolecular rate constant k2 of 1.33 10 m 1 s 1 for the dioxygen activation of the CuCuCu 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. The process can be made catalytic by adding the appropriate amounts of H2O2 to regenerate the spent catalyst after O-atom transfer from the activated tricopper complex to CH4. This multiple-turnover reaction is depicted in Figure 1C. In these experiments, the [CuCuCu(7-N-Etppz)] catalyst is activated by O2 as in the single-turnover experiment described earlier, but the spent catalyst is regenerated by twoelectron reduction by a molecule of H2O2 (Figure 2A). Because the effective turnover number (TON), or the total equivalent of products formed over the time course of the experiment, peaks at approximately six when the turnover is initiated with 20 equivalents of H2O2, it is evident that abortive cycling begins to kick in when the steady-state concentration of the H2O2 concentration exceeds approximately ten equivalents. When the steady-state H2O2 concentration is above this level, reductive abortion of the activated catalyst becomes competitive with the O-atom transfer to methane to produce methanol. In this case, the rate of O-atom transfer is limited by the relatively low solubility of CH4 in ACN under ambient conditions of temperature and pressure (Figure 2B). The [CuCuCu(7-N-Etppz)] complex also mediates the catalytic oxidation of normal C2–C6 alkanes (data not shown) [*] Prof. Dr. S. I. Chan, Y.-J. Lu, Dr. P. Nagababu, Dr. S. Maji, M.-C. Hung, P. D. Minh, J. C.-H. Lai, K. Y. Ng, Prof. Dr. S. S.-F. Yu Institute of Chemistry, Academia Sinica Nankang, Taipei 11529 (Taiwan) E-mail: sunneychan@yahoo.com sfyu@gate.sinica.edu.tw

Quantitative Proteomic Analysis of Metabolic Regulation by Copper Ions in Methylococcus capsulatus (Bath)

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 C1 metabolism pathways in this methanotroph and clarified the interrelationships between them.

Steric Zipper of the Amyloid Fibrils Formed by Residues 109–122 of the Syrian Hamster Prion Protein

We report the results of atomic force microscopy, Fourier-transform infrared spectroscopy, solid-state nuclear magnetic resonance, and molecular dynamics (MD) calculations for amyloid fibrils formed by residues 109-122 of the Syrian hamster prion protein (H1). Our data reveal that H1 fibrils contain no more than two beta-sheet layers. The peptide strands of H1 fibrils are antiparallel with the A117 residues aligned to form a linear chain in the direction of the fibril axis. The molecular structure of the H1 fibrils, which adopts the motif of steric zipper, is highly uniform in the region of the palindrome sequence AGAAAAGA. The closest distance between the two adjacent beta-sheet layers is found to be about 5 A. The structural features of the molecular model of H1 fibrils obtained by MD simulations are consistent with the experimental results. Overall, our solid-state NMR and MD simulation data indicate that a steric zipper, which was first observed in the crystals of fibril-forming peptides, can be formed in H1 fibrils near the region of the palindrome sequence.

Adsorption of a Statherin Peptide Fragment on the Surface of Nanocrystallites of Hydroxyapatite

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.

Alkane Oxidation: Methane Monooxygenases, Related Enzymes, and Their Biomimetics

Methane monooxygenases (MMOs) mediate the facile conversion of methane into methanol in methanotrophic bacteria with high efficiency under ambient conditions. Because the selective oxidation of methane is extremely challenging, there is considerable interest in understanding how these enzymes carry out this difficult chemistry. The impetus of these efforts is to learn from the microbes to develop a biomimetic catalyst to accomplish the same chemical transformation. Here, we review the progress made over the past two to three decades toward delineating the structures and functions of the catalytic sites in two MMOs: soluble methane monooxygenase (sMMO) and particulate methane monooxygenase (pMMO). sMMO is a water-soluble three-component protein complex consisting of a hydroxylase with a nonheme diiron catalytic site; pMMO is a membrane-bound metalloenzyme with a unique tricopper cluster as the site of hydroxylation. The metal cluster in each of these MMOs harnesses O2 to functionalize the C-H bond using different chemistry. We highlight some of the common basic principles that they share. Finally, the development of functional models of the catalytic sites of MMOs is described. These efforts have culminated in the first successful biomimetic catalyst capable of efficient methane oxidation without overoxidation at room temperature.

Bacterial siderophores: the solution stoichiometry and coordination of the Fe(III) complexes of pyochelin and related compounds.

Pyochelin, its analog 3′′-nor-NH-pyochelin, and the related methyl hydroxamate, 2-(2′-hydroxyphenyl)-4,5-dihydrothiazol-4-carboxylic acid methoxymethyl amide, have been prepared together with their Fe(III) complexes. The solution stoichiometry and the coordination of the three Fe(III) complexes in methanol or buffered (pH∼2) 50:50 (v/v) methanol–water mixtures were determined using various spectroscopic methods: UV–vis absorption, X-ray absorption, extended X-ray absorption fine structure and electron paramagnetic resonance. All three systems showed both a 1:1 and 2:1 ligand–Fe(III) stoichiometry, but presented different coordination properties. Conditional formation constants (pH∼2) were determined for both the 1:1 and 2:1 complexes in all three systems. Computation of the coordination-conformational energies by semiempirical methods indicated that the coordination in the case of the 2:1 complexes of pyochelin–Fe(III) and 3′′-nor-NH-pyochelin–Fe(III) was asymmetrical, with one molecule of pyochelin (or 3′′-nor-NH-pyochelin) tetradentately coordinated (O1, N1, N2 and O3) to the Fe(III), and the second molecule bound bidentately (O1, N1 or N2, O3), to complete the octahedral geometry. In contrast, two molecules of the methyl hydroxamate each provided a set of tridentate ligand atoms in the formation of the 2:1 ligand–Fe(III) complex. These results are consistent with the role of pyochelin in the uptake of iron by the FptA receptor in the outer membrane of Pseudomonas aeruginosa and in several gram-negative bacteria.

The stereospecific hydroxylation of [2,2-2H2]butane and chiral dideuteriobutanes by the particulate methane monooxygenase from Methylococcus capsulatus (Bath).

Experiments on cryptically chiral ethanes have indicated that the particulate methane monooxygenase (pMMO) from Methylococcus capsulatus (Bath) catalyzes the hydroxylation of ethane with total retention of configuration at the carbon center attacked. This result would seem to rule out a radical mechanism for the hydroxylation chemistry, at least as mediated by this enzyme. The interpretation of subsequent experiments on n-propane, n-butane, and n-pentane has been complicated by hydroxylation at both the pro-R and pro-S secondary C–H bonds, where the hydroxylation takes place. It has been suggested that these results merely reflect presentation of both the pro-R and pro-S C–H bonds to the hot “oxygen atom” species generated at the active site, and that the oxo-transfer chemistry, in fact, proceeds concertedly with retention of configuration. In the present work, we have augmented these earlier studies with experiments on [2,2-2H2]butane and designed d,l form chiral dideuteriobutanes. Essentially equal amounts of (2R)-[3,3-2H2]butan-2-ol and (2R)-[2-2H1]butan-2-ol are produced upon hydroxylation of [2,2-2H2]butane. The chemistry is stereospecific with full retention of configuration at the secondary carbon oxidized. In the case of the various chiral deuterated butanes, the extent of configurational inversion has been shown to be negligible for all the chiral butanes examined. Thus, the hydroxylation of butane takes place with full retention of configuration in butane as well as in the case of ethane. These results are interpreted in terms of an oxo-transfer mechanism based on side-on singlet oxene insertion across the C–H bond similar to that previously noted for singlet carbene insertion (Kirmse, W., and Özkir, I. S. (1992) J. Am. Chem. Soc. 114, 7590-7591). Finally, we discuss how even the oxene insertion mechanism, with “spin crossover” in the transition state, could lead to small amounts of radical rearrangement products, if and when such products are observed. A scheme is described that unifies the two extreme mechanistic limits, namely the concerted oxene insertion and the hydrogen abstraction radical rebound mechanism within the same over-arching framework.

Probing the Hydrophobic Pocket of the Active Site in the Particulate Methane Monooxygenase (pMMO) from Methylococcus capsulatus (Bath) by Variable Stereoselective Alkane Hydroxylation and Olefin Epoxidation

pMMO from M. capsulatus (Bath) oxidizes straight‐chain C1–C5 alkanes and alkenes to form their corresponding 2‐alcohols and epoxides. According to experiments performed with cryptically chiral ethane and D,L‐[2‐2H1,3‐2H1]butane, the reactions proceed through the concerted O‐atom insertion mechanism. However, when propene and but‐1‐ene are used as epoxidation substrates, the enantiomeric excesses (ees) of the enzymatic products are only 18 and 37 %, respectively. This relatively poor stereoselectivity in the enzymatic epoxidation presumably reflects low stereochemical differentiation between the re and si faces in the hydrophobic pocket of the active site. Further insights into the reaction mechanism are now provided by studies on trans‐but‐2‐ene, which reveal only the D,L‐2,3‐dimethyloxirane products, and on cis‐but‐2‐ene, which yield only the meso product. These observations indicate that the enzymatic epoxidation indeed proceeds through electrophilic syn addition. To achieve better facial selectivity, we have also used 3,3,3‐trifluoroprop‐1‐ene as the substrate. The products obtained are 90 % (2S)‐oxirane. When 1,1,1‐trifluoropropane is the substrate, the hydroxylation at the 2‐carbon exhibits an inverse chiral selectivity relative to that seen with normal butane, if we consider the size of the CF3 group in the fluorinated propane to be comparable to one of the ethyl groups in butane. These experiments are beginning to delineate the factors that influence the orientations of various substrates in the hydrophobic cavity of the active site in the enzyme.

Isolation, purification and characterization of hemerythrin from Methylococcus capsulatus (Bath)

Earlier work from our laboratory has indicated that a hemerythrin-like protein was over-produced together with the particulate methane monooxygenase (pMMO) when Methylococcus capsulatus (Bath) was grown under high copper concentrations. A homologue of hemerythrin had not previously been found in any prokaryote. To confirm its identity as a hemerythrin, we have isolated and purified this protein by ion-exchange, gel-filtration and hydrophobic interaction chromatography, and characterized it by mass spectrometry, UV-visible, CD, EPR and resonance Raman spectroscopy. On the basis of biophysical and multiple sequence alignment analysis, the protein isolated from M. capsulatus (Bath) is in accord with hemerythrins previously reported from higher organisms. Determination of the Fe content in conjunction with molecular-weight estimation and mass analysis indicates that the native hemerythrin in M. capsulatus (Bath) is a monomer with molecular mass 14.8 kDa, in contrast to hemerythrins from other eukaryotic organisms, where they typically exist as a tetramer or higher oligomers.