We report CW and pulsed Q-band 1,2H ENDOR measurements of intermediate X formed during the assembly of the diferric tyrosyl radical cofactor of the R2 subunit in ribonucleotide reductase. These studies, performed with H2O and D2O buffers, were designed to determine whether the exchangeable proton signals are associated with an hydroxo bridge, a terminal water, or both. In doing so, we identify the types of protonated oxygen (OH x ) species coordinated to the iron ions of X and their disposition relative to the ferric and ferryl iron ions. The exchangeable proton signals displayed by intermediate X belong to two protons associated with a terminal water bound to Fe,III and not to an hydroxo bridge; within the precision of the modeling, this picture of a terminal water is indistinguishable from that of a 2-fold disordered terminal hydroxyl. The fact that X displays strong spin-coupling between iron ions requires that there be one or more oxo/hydroxo bridges. These findings then establish that X contains the [(H x O)FeIIIOFeIV] fragment.

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 ...

N2 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 (E3 or E4 states), with the E4 state being optimally activated. Here we study the FeMo-co 57Fe atoms of E4 trapped with the α-70Val→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 57Fe hyperfine couplings for five of the seven iron sites of the reductively activated E4 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 57Fe measurements yield both the magnitude and signs of the isotropic couplings for the complete set of seven Fe sites of FeMo-co in E4. In light of the previous findings that FeMo-co of E4 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 in E4 to that of the resting state (MN), with the four accumulated electrons residing on the two Fe-bound hydrides. Comparisons with earlier 57Fe 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 N2 plus 2H+ to two NH3 plus H2, the inorganic core of FeMo-co cycles through only a single redox couple connecting the two formal redox levels: those associated with the resting state, MN, and the one-electron reduced state, MR. We further note that this conjecture might apply to other complex FeS enzymes.

The fully oxidized state of cytochrome c peroxidase (CcP), called ES, contains two oxidizing equivalents, one as an oxyferryl heme and the other as an organic radical on an amino acid residue. The unusual electron paramagnetic resonance spectrum of ES has been shown to be due to a weak, distributed exchange coupling between the two paramagnetic redox centers (Houseman, A.