Ligand spin densities in blue copper proteins by q-band proton and nitrogen-14 ENDOR spectroscopy

Self Cite

114

Nitrogenase catalyzes the reduction of N 2 and protons to yield two NH 3 and one H 2 . Substrate binding occurs at a complex organo-metallocluster called FeMo-cofactor (FeMo-co). Each catalytic cycle involves the sequential delivery of eight electrons/protons to this cluster, and this process has been framed within a kinetic scheme developed by Lowe and Thorneley. Rapid freezing of a modified nitrogenase under turnover conditions using diazene, methyldiazene (HN = N-CH 3 ), or hydrazine as substrate recently was shown to trap a common intermediate, designated I . It was further concluded that the two N-atoms of N 2 are hydrogenated alternately (“Alternating” ( A ) pathway). In the present work, Q-band CW EPR and 95 Mo ESEEM spectroscopy reveal such samples also contain a common intermediate with FeMo-co in an integer-spin state having a ground-state “non-Kramers” doublet. This species, designated H , has been characterized by ESEEM spectroscopy using a combination of 14,15 N isotopologs plus 1,2 H isotopologs of methyldiazene. It is concluded that: H has NH 2 bound to FeMo-co and corresponds to the penultimate intermediate of N 2 hydrogenation, the state formed after the accumulation of seven electrons/protons and the release of the first NH 3 ; I corresponds to the final intermediate in N 2 reduction, the state formed after accumulation of eight electrons/protons, with NH 3 still bound to FeMo-co prior to release and regeneration of resting-state FeMo-co. A proposed unification of the Lowe-Thorneley kinetic model with the “prompt” alternating reaction pathway represents a draft mechanism for N 2 reduction by nitrogenase.

Section: Methodsmentioning

confidence: 99%

Unification of reaction pathway and kinetic scheme for N ₂ reduction catalyzed by nitrogenase

Lukoyanov

Yang

Barney

et al. 2012

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114

“…To remove Hepes, which interferes with CD signals at short wavelengths, pMMO was exchanged twice into a 0.1% 12DM solution in ddH 2 O by using a Microcon 100 (Millipore). X-band EPR spectra were acquired at 120 K by using a modified Varian E-4 spectrometer; Q-band EPR spectra were acquired at 2 K by using an instrument as described (33). EPR samples of membrane-bound and purified pMMO were frozen in liquid nitrogen.…”

mentioning

confidence: 99%

Purified particulate methane monooxygenase from Methylococcus capsulatus (Bath) is a dimer with both mononuclear copper and a copper-containing cluster

Lieberman

Shrestha

Doan

et al. 2003

Self Cite

146

207

Particulate methane monooxygenase (pMMO) is a membranebound enzyme that catalyzes the oxidation of methane to methanol in methanotropic bacteria. Understanding how this enzyme hydroxylates methane at ambient temperature and pressure is of fundamental chemical and potential commercial importance. Difficulties in solubilizing and purifying active pMMO have led to conflicting reports regarding its biochemical and biophysical properties, however. We have purified pMMO from Methylococcus capsulatus (Bath) and detected activity. The purified enzyme has a molecular mass of Ϸ200 kDa, probably corresponding to an ␣2␤2␥2 polypeptide arrangement. Each 200-kDa pMMO complex contains 4.8 ؎ 0.8 copper ions and 1.5 ؎ 0.7 iron ions. Electron paramagnetic resonance spectroscopic parameters corresponding to 40 -60% of the total copper are consistent with the presence of a mononuclear type 2 copper site. X-ray absorption near edge spectra indicate that purified pMMO is a mixture of Cu(I) and Cu(II) oxidation states. Finally, extended x-ray absorption fine structure data are best fit with oxygen͞nitrogen ligands and a 2.57-Å Cu-Cu interaction, providing direct evidence for a copper-containing cluster in pMMO. O ne of the great challenges for the chemical and engineering communities is the selective oxidation of methane to methanol. Although world reserves of petroleum and natural gas are comparable, methane is used far less efficiently as an energy source because it has a low energy density and is hazardous and expensive to transport (1). Conversion of methane to a liquid such as methanol would solve this problem, but current industrial processes for this transformation are costly and inefficient, requiring high temperatures and pressures (2, 3). By contrast, methanotrophic bacteria (4) oxidize methane to methanol at ambient temperature and pressure by using methane monooxygenase (MMO) enzyme systems (5). Only one other enzyme, ammonia monooxygenase (6), can activate the COH bond in methane (104 kcal͞mol). Cytochromes P450 (7) and copper monooxygenases (8) oxidize larger, more reactive hydrocarbons, but cannot hydroxylate methane. Therefore, a detailed understanding of biological methane oxidation is of both fundamental chemical and potential commercial importance.All methanotrophs produce a membrane-bound MMO called particulate MMO (pMMO) (5). Under conditions of low copper availability, several strains also express a soluble enzyme (sMMO) (9), which contains a catalytic diiron center (10). Whereas the structure, biochemistry, and mechanism of sMMO are well understood (11), studies of pMMO are less advanced because of difficulties in solubilizing and purifying active enzyme. The Methylococcus capsulatus (Bath) pMMO comprises three polypeptides, the ␣ (Ϸ47 kDa), ␤ (Ϸ24 kDa), and ␥ (Ϸ22 kDa) subunits, encoded by the pmoB, pmoA, and pmoC genes, respectively (12, 13). It is not known how these three polypeptides are arranged in the pMMO holoenzyme. According to radiolabeling experiments with the suicide substrate acetylene, the active ...

“…The electronic structure of the blue copper site of plastocyanin has been studied extensively during the last decades both by experimental approaches (5,16,17,38) and quantum chemical calculations (5,10,(25)(26)(27)47). It was found that the unpaired electron spin density is located predominately at the atomic orbitals of the four atoms in the trigonal NNS-plane, i.e., the copper atom, the Cys S ␥ ligand, and the two aromatic His N ␦ ligands (5,16,17,(25)(26)(27)30), whereas the electron spin density at the Met S ␦ atom and the Cys H ␤ is small (26)(27)(28)38). Yet, it was found recently (31) that also the latter orbitals must be included in the description of the unpaired electron spin density to obtain a reliable prediction of the paramagnetic relaxation of the protons close to the copper site.…”

Section: Distribution Of the Unpaired Electron Spin In Plastocyaninmentioning

confidence: 99%

“…Because the paramagnetic restraints are derived from the relaxation rates of nuclei close to the metal site, the point dipole approximation of the electron spin fails and a description of the paramagnetic relaxation that takes the electron delocalization into account must be used (37). Therefore, the approach requires knowledge of the distribution of the unpaired electron spin, which can be obtained experimentally by x-ray absorption spectroscopy (16,17), electron nuclear double resonance (38), paramagnetic NMR (28-31) spectroscopy, or theoretically by quantum chemical calculations (5,(25)(26)(27). The blue copper protein, plastocyanin from Anabaena variabilis (A.v.…”

mentioning

confidence: 99%

Determination of the geometric structure of the metal site in a blue copper protein by paramagnetic NMR

Hansen

Led

2006

The biological function of metalloproteins is closely tied to the geometric and electronic structures of the metal sites. Here, we show that the geometric structure of the metal site of a metalloprotein in solution can be determined from experimentally measured electron-nuclear spin-spin interactions obtained by NMR. Thus, the geometric metal site structure of plastocyanin from Anabaena variabilis was determined by including the paramagnetic relaxation enhancement of protons close to the copper site as restraints in a conventional NMR structure determination, together with the distribution of the unpaired electron onto the ligand atoms. Also, the interproton distances (nuclear Overhauser enhancements) and dihedral angles (scalar nuclear spin-spin couplings) normally used in NMR structure determinations were included as restraints. The structure calculations were carried out with the program X-PLOR and a module that takes into account the specific characteristics of the paramagnetic restraints. A well defined metal site structure was obtained with the structural characteristics of the blue copper site, including a distorted tetrahedral geometry, a short Cu-Cys S ␥ bond, and a long Cu-Met S ␦ bond. Overall, the agreement of the obtained metal site structure of Anabaena variabilis plastocyanin with those of other plastocyanins obtained by x-ray crystallography confirms the reliability of the approach.metal site structure ͉ metalloproteins ͉ paramagnetic nuclear relaxation T he biological function of many metalloproteins stems from the geometric and electronic structures of the metal sites imposed by the protein environment. In the blue copper proteins, such as plastocyanins, amicyanins, and azurins, the geometry of the copper site is unusual as compared with smallmolecule copper complexes (1-15). In particular, the metal sites of blue copper proteins are characterized by a short coppersulfur bond. This unusual geometry is believed to be the main reason for the strong covalency of the metal site (10,16,17) and, thus, responsible for the rapid and long-range electron transfer reactivity (18-22) that characterizes the blue copper proteins. Detailed knowledge of the geometric and electronic metal site structures of the blue copper proteins is, therefore, imperative for understanding the function of the proteins at the molecular level.So far, the geometric structure of the metal site in blue copper proteins (12-14) has been determined primarily by x-ray crystallography and extended x-ray absorption fine structure (3,23,24), whereas the electronic structure of the blue copper site has been determined theoretically from quantum chemical calculations (5, 25-27) and experimentally by x-ray absorption spectroscopy (16, 17), and more recently by nuclear paramagnetic relaxation (28-31). These structures have formed the basis for a detailed understanding of the biological function of the proteins by elucidating the interplay between the electronic and geometric structure of the metal site (8,15,(32)(33)(34). However, the geomet...