Joshua Telser

The metal centers of particulate methane monooxygenase from Methylosinus trichosporium OB3b.

Particulate methane monooxygenase (pMMO) is a membrane-bound metalloenzyme that oxidizes methane to methanol in methanotrophic bacteria. The nature of the pMMO active site and the overall metal content are controversial, with spectroscopic and crystallographic data suggesting the presence of a mononuclear copper center, a dinuclear copper center, a trinuclear center, and a diiron center or combinations thereof. Most studies have focused on pMMO from Methylococcus capsulatus (Bath). pMMO from a second organism, Methylosinus trichosporium OB3b, has been purified and characterized by spectroscopic and crystallographic methods. Purified M. trichosporium OB3b pMMO contains approximately 2 copper ions per 100 kDa protomer. Electron paramagnetic resonance (EPR) spectroscopic parameters indicate that type 2 Cu(II) is present as two distinct species. Extended X-ray absorption fine structure (EXAFS) data are best fit with oxygen/nitrogen ligands and reveal a Cu-Cu interaction at 2.52 A. Correspondingly, X-ray crystallography of M. trichosporium OB3b pMMO shows a dinuclear copper center, similar to that observed previously in the crystal structure of M. capsulatus (Bath) pMMO. There are, however, significant differences between the pMMO structures from the two organisms. A mononuclear copper center present in M. capsulatus (Bath) pMMO is absent in M. trichosporium OB3b pMMO, whereas a metal center occupied by zinc in the M. capsulatus (Bath) pMMO structure is occupied by copper in M. trichosporium OB3b pMMO. These findings extend previous work on pMMO from M. capsulatus (Bath) and provide new insight into the functional importance of the different metal centers.

Crystal Structure and Characterization of Particulate Methane Monooxygenase from Methylocystis species Strain M

Particulate methane monooxygenase (pMMO) is an integral membrane metalloenzyme that oxidizes methane to methanol in methanotrophic bacteria. Previous biochemical and structural studies of pMMO have focused on preparations from Methylococcus capsulatus (Bath) and Methylosinus trichosporium OB3b. A pMMO from a third organism, Methylocystis species strain M, has been isolated and characterized. Both membrane-bound and solubilized Methylocystis sp. strain M pMMO contain ~2 copper ions per 100 kDa protomer and exhibit copper-dependent propylene epoxidation activity. Spectroscopic data indicate that Methylocystis sp. strain M pMMO contains a mixture of Cu(I) and Cu(II), of which the latter exhibits two distinct type 2 Cu(II) electron paramagnetic resonance (EPR) signals. Extended X-ray absorption fine structure (EXAFS) data are best fit with a mixture of Cu-O/N and Cu-Cu ligand environments with a Cu-Cu interaction at 2.52-2.64 Å. The crystal structure of Methylocystis sp. strain M pMMO was determined to 2.68 Å resolution and is the best quality pMMO structure obtained to date. It provides a revised model for the pmoA and pmoC subunits and has led to an improved model of M. capsulatus (Bath) pMMO. In these new structures, the intramembrane zinc/copper binding site has a different coordination environment from that in previous models.

Evidence for N coordination to Fe in the [2Fe-2S] center in yeast mitochondrial complex III Comparison with similar findings for analogous bacterial [2Fe-2S] proteins

Yeast mitochondrial complex III contains a subunit with a [2Fe‐2S] cluster (the Rieske center) that has unusual physical and chemical properties. For apparently similar centers isolated from bacteria, it has been shown by electron nuclear double resonance (ENDOR) and electron spin echo envelope modulation (ESEEM) measurements that these [2Fe‐2S] centers are coordinated by at least one and probably two nitrogen ligands. This work describes similar ENDOR and ESEEM studies on the intact mitochondrial complex. We find that this [2Fe‐2S] cluster exhibits ESEEM and ENDOR properties that appear to be indistinguishable from those observed with the isolated bacterial systems. Furthermore, changes in EPR lineshape that occur as complex III is progressively reduced are not accompanied by any changes in the nitrogen coupling parameters. This spectroscopic evidence for nitrogen coordination is supported by published sequence data on four Rieske iron‐sulfur subunits. It seems likely that this is a general characteristic of such [2Fe‐2S] redox active centers.

57Fe ENDOR Spectroscopy and ‘Electron Inventory’ Analysis of the Nitrogenase E4 Intermediate Suggest the Metal-Ion Core of FeMo-cofactor Cycles Through Only One Redox Couple

N(2) binds to the active-site metal cluster in the nitrogenase MoFe protein, the FeMo-cofactor ([7Fe-9S-Mo-homocitrate-X]; FeMo-co) only after the MoFe protein has accumulated three or four electrons/protons (E(3) or E(4) states), with the E(4) state being optimally activated. Here we study the FeMo-co (57)Fe atoms of E(4) trapped with the α-70(Val→Ile) MoFe protein variant through use of advanced ENDOR methods: random-hop Davies pulsed 35 GHz ENDOR; difference triple resonance; the recently developed Pulse-Endor-SaTuration and REcovery (PESTRE) protocol for determining hyperfine-coupling signs; and Raw-DATA (RD)-PESTRE, a PESTRE variant that gives a continuous sign readout over a selected radiofrequency range. These methods have allowed experimental determination of the signed isotropic (57)Fe hyperfine couplings for five of the seven iron sites of the reductively activated E(4) FeMo-co, and given the magnitude of the coupling for a sixth. When supplemented by the use of sum-rules developed to describe electron-spin coupling in FeS proteins, these (57)Fe measurements yield both the magnitude and signs of the isotropic couplings for the complete set of seven Fe sites of FeMo-co in E(4). In light of the previous findings that FeMo-co of E(4) binds two hydrides in the form of (Fe-(μ-H(-))-Fe) fragments, and that molybdenum has not become reduced, an electron inventory analysis assigns the formal redox level of FeMo-co metal ions in E(4) to that of the resting state (M(N)), with the four accumulated electrons residing on the two Fe-bound hydrides. Comparisons with earlier (57)Fe ENDOR studies and electron inventory analyses of the bio-organometallic intermediate formed during the reduction of alkynes and the CO-inhibited forms of nitrogenase (hi-CO and lo-CO) inspire the conjecture that throughout the eight-electron reduction of N(2) plus 2H(+) to two NH(3) plus H(2), the inorganic core of FeMo-co cycles through only a single redox couple connecting two formal redox levels: those associated with the resting state, M(N), and with the one-electron reduced state, M(R). We further note that this conjecture might apply to other complex FeS enzymes.

Metalloenzyme Active-Site Structure and Function through Multifrequency CW and Pulsed ENDOR

Electron paramagnetic resonance (EPR) techniques have long been major tools in efforts to determine the structure and function of metalloen-zyme active sites (Beinert et al., 1962; Hoff, 1989). Much of the information EPR provides about the composition, structure, and bonding of a paramagnetic metal center is obtained by analysis of hyperfine coupling constants (Abragam and Bleaney, 1970; Atherton, 1973) that arise from interactions between the spin of the unpaired electron(s) and the spins of nuclei associated with the metal center, endogenous ligands, or bound substrate. At the most basic level, the observation of hyperfine coupling and its assignment to one or more nuclei (e.g., 1H, 14N) provide information about the chemical composition of the center. Detailed analysis of these couplings can provide information about its geometry or about substrate binding, as well as deep insights into chemical bonding. In principle these coupling constants can be calculated from splittings in the EPR spectrum. However, as illustrated in Fig. 1, for most metalloproteins these splittings cannot be resolved, and thus the chemical information they carry is lost.

Observation of organometallic and radical intermediates formed during the reaction of methyl-coenzyme M reductase with bromoethanesulfonate.

Methyl-coenzyme M reductase (MCR) from methanogenic archaea catalyzes the final step of methane formation, in which methyl-coenzyme M (2-methylthioethanesulfonate, methyl-SCoM) is reduced with coenzyme B (N-(7-mercaptoheptanoyl)threonine phosphate, CoBSH) to form methane and the heterodisulfide CoBS-SCoM. The active dimeric form of MCR contains two Ni(I)-F(430) prosthetic groups, one in each monomer. This report describes studies of the reaction of the active Ni(I) state of MCR (MCR(red1)) with BES (2-bromoethanesulfonate) and CoBSH or its analogue, CoB(6)SH (N-(6-mercaptohexanoyl)threonine phosphate), by transient kinetic measurements using EPR and UV-visible spectroscopy and by global fits of the data. This reaction is shown to lead to the formation of three intermediates, the first of which is assigned as an alkyl-Ni(III) species that forms as the active Ni(I)-MCR(red1) state of the enzyme decays. Subsequently, a radical (MCR(BES) radical) is formed that was characterized by multifrequency electron paramagnetic resonance (EPR) studies at X- ( approximately 9 GHz), Q- ( approximately 35 GHz), and D- ( approximately 130 GHz) bands and by electron-nuclear double resonance (ENDOR) spectroscopy. The MCR(BES) radical is characterized by g-values at 2.00340 and 1.99832 and includes a strongly coupled nonexchangeable proton with a hyperfine coupling constant of 50 MHz. Based on transient kinetic measurements, the formation and decay of the radical coincide with a species that exhibits absorption peaks at 426 and 575 nm. Isotopic substitution, multifrequency EPR, and ENDOR spectroscopic experiments rule out the possibility that MCR(BES) is a tyrosyl radical and indicate that if a tyrosyl radical is formed during the reaction, it does not accumulate to detectable levels. The results provide support for a hybrid mechanism of methanogenesis by MCR that includes both alkyl-Ni and radical intermediates.