The electronic and redox properties of the iron and tungsten centers in the aldehyde ferredoxin oxidoreductases (AORs) from Pyrococcus furiosus (Pf) and Pyrococcus strain ES-4 (ES-4) have been investigated by the combination of EPR and variable-temperature magnetic circular dichroism (VTMCD) spectroscopies. Parallel- and perpendicular-mode EPR studies of ES-4 AOR reveal a redox inactive “g = 16” resonance from an integer spin paramagnet. On the basis of the X-ray crystal structure of Pf AOR (Chan, M. K.; Mukund, S.; Kletzin, A.; Adams, M. W. W.; Rees, D. C. Science 1995, 267, 1463−1469), this resonance is attributed to a mononuclear high-spin Fe(II) ion at the subunit interface, although the possibility that this center is a carboxylate-bridged reduced diiron center in ES-4 AOR is also considered. Both enzymes have a [4Fe−4S]2+,+ cluster with unique electronic properties compared to known synthetic or biological [4Fe−4S]+ clusters, i.e. pure S = 3/2 ground state with g = 4.7, 3.4, 1.9 (E/D = 0.12 and D = +4 cm-1). Seven distinct W(V) EPR signals have been observed during dye-mediated redox titrations of Pf AOR, and the four major W(V) species have been rigorously identified and characterized via EPR spectral simulations of natural abundance and 183W-enriched samples (183W, I = 1/2, 14.28% natural abundance). Both enzymes contain two major forms of W, each corresponding to approximately 20−30% of the total W. One of these is a catalytically competent W species that cycles between the W(IV)/W(V)/W(VI) states at physiologically relevant potentials (<−300 mV) and gives rise to the “low-potential” W(V) resonance, g ∼ 1.99, 1.90, 1.86. This form of W is quantitatively and irreversibly converted into a distinct and inactive W(IV)/W(V) species by the addition of high concentrations of glycerol or ethylene glycol at 80 °C and is responsible for the “diol-inhibited” W(V) resonance, g ∼ 1.96, 1.94, 1.89. The other major form of W gives rise to a “high-potential” W(V) species, g ∼ 1.99, 1.96, 1.89, at nonphysiologically relevant potentials (>0 mV), as a result of a one-electron redox process that is tentatively attributed to ligand based oxidation of a W(VI) species. In addition, active samples of Pf AOR, in particular, can have up to 20% of the W as an inactive W(VI)/W(V) species, with a midpoint potential close to −450 mV, and is responsible for the “spin-coupled” W(V) resonance. This W(V) signal exhibits a broad complex resonance spanning 600 mT due to weak spin−spin interaction with the nearby S = 3/2 [4Fe−4S]+ cluster. Structures are proposed for each of the major W(V) species on the basis of EPR g values and 183W A values as compared to other biological and synthetic W(V)/Mo(V) centers, VTMCD spectra, and the available X-ray crystallographic and XAS data for Pf AOR and the Mo-containing DMSO reductase from Rhodobacter sphaeroides. Comparison with the limited spectroscopic data that are available for all known tungstoenzymes suggests two major classes of enzyme with distinct active site structures.

A mutant form of Klebsiella aerogenes urease possessing Ala instead of His at position 134 (H134A) is inactive and binds approximately half the normal complement of nickel (Park, I.-S., and Hausinger, R. P. (1993) Protein Sci. 2, 1034 -1041). The crystal structure of the H134A protein was obtained at 2.0-Å resolution, and it confirms that only Ni-1 of the two nickel ions found in the native enzyme is present. In contrast to the pseudotetrahedral geometry observed for Ni-1 in native urease (where it is liganded by His-246, His-272, one oxygen atom of carbamylated Lys-217, and a water molecule at partial occupancy), the mononickel metallocenter in the H134A protein was found to possess octahedral geometry and was coordinated by the above protein ligands plus three water molecules. The nickel site of H134A urease was probed by UV-visible, variable temperature magnetic circular dichroism, and x-ray absorption spectroscopies. The spectroscopic data are consistent with the presence of Ni(II) in octahedral geometry coordinated by two histidylimidazoles and additional oxygen and/or nitrogen donors. These data underscore the requirement of Ni-2 for formation of active urease and demonstrate the important role of Ni-2 in establishing the proper Ni-1 coordination geometry.Urease (EC 3.5.1.5) is a nickel-containing enzyme that catalyzes the hydrolysis of urea (1, 2). In addition to playing a key role in plant (3) and microbial (4) nitrogen metabolism, the enzyme has been implicated as a virulence factor in various human and animal pathogens (reviewed in Ref.2). The threedimensional structure has been resolved to 2.2 Å (5) for the best characterized urease, that from the enteric bacterium Klebsiella aerogenes. The protein is a trimer of trimers ((␣␤␥) 3 ) composed of subunits with M r ϭ 60,304 (␣), 11,695 (␤), and 11,086 (␥). The enzyme active site is located in the ␣-subunit and contains a binickel center in which the two metal ions are separated by 3.5 Å and bridged by carbamylated Lys-217. In the crystallographic model, one nickel ion (Ni-2) exhibits distorted trigonal bipyramidal or distorted square pyramidal geometry in which two nitrogen ligands are derived from His-134 and His-136, and three oxygen atoms are contributed by carbamylated Lys-217, Asp-360, and a solvent molecule (Wat-1). 1The second urease metal ion (Ni-1) exhibits pseudotetrahedral geometry, a coordination that is unusual for nickel. The ligands to Ni-1 include the second oxygen atom of the carbamylated Lys-217, nitrogen atoms from His-246 and His-272, and partial coordination to the solvent molecule that is strongly coordinated to Ni-2.In an effort to better characterize the novel coordination geometry observed in the Ni-1 site of urease, we have examined a mutant with His-134 substituted by Ala (H134A) that contains only this metal ion (6). We compare the crystal structure and the UV-visible, XAS, and VTMCD spectroscopic signatures of the H134A protein to the corresponding properties of the native enzyme (5, 7-9).2 We demonstrate that the Ni-1 metallo...

The electronic and redox properties of the iron-sulfur cluster and tungsten center in the as-isolated and sulfide-activated forms of formaldehyde ferredoxin oxidoreductase (FOR) from Thermococcus litoralis (Tl) have been investigated by using the combination of EPR and variable-temperature magnetic circular dichroism (VTMCD) spectroscopies. The results reveal a [Fe4S4]2+,+ cluster (Em=-368mV) that undergoes redox cycling between an oxidized form with an S=0 ground state and a reduced form that exists as a pH- and medium-dependent mixture of S=3/2 (g=5.4; E/D=0.33) and S=1/2 (g=2.03, 1.93, 1.86) ground states, with the former dominating in the presence of 50% (v/v) glycerol. Three distinct types of W(V) EPR signals have been observed during dye-mediated redox titration of as-isolated Tl FOR. The initial resonance observed upon oxidation, termed the "low-potential" W(V) species (g=1.977, 1.898, 1.843), corresponds to approximately 25-30% of the total W and undergoes redox cycling between W(IV)/ W(V) and W(V)/W(VI) states at physiologically relevant potentials (Em= -335 and -280 mV, respectively). At higher potentials a minor "mid-potential" W(V) species, g= 1.983, 1.956, 1.932, accounting for less than 5 % of the total W, appears with a midpoint potential of -34 mV and persists up to at least + 300 mV. At potentials above 0 mV, a major "high-potential" W(V) signal, g= 1.981, 1.956, 1.883, accounting for 30-40% of the total W, appears at a midpoint potential of +184 mV. As-isolated samples of Tl FOR were found to undergo an approximately 8-fold enhancement in activity on incubation with excess Na2S under reducing conditions and the sulfide-activated Tl FOR was partially inactivated by cyanide. The spectroscopic and redox properties of the sulfide-activated Tl FOR are quite distinct from those of the as-isolated enzyme, with loss of the low-potential species and changes in both the mid-potential W(V) species (g= 1.981, 1.950, 1.931; Em = -265 mV) and high-potential W(V) species (g=1.981, 1.952, 1.895; Em = +65 mV). Taken together, the W(V) species in sulfide-activated samples of Tl FOR maximally account for only 15% of the total W. Both types of high-potential W(V) species were lost upon incubation with cyanide and the sulfide-activated high-potential species is converted into the as-isolated high-potential species upon exposure to air. Structural models are proposed for each of the observed W(V) species and both types of mid-potential and high-potential species are proposed to be artifacts of ligand-based oxidation of W(VI) species. A W(VI) species with terminal sulfido or thiol ligands is proposed to be responsible for the catalytic activity in sulfide-activated samples of Tl FOR.

A strong desire to upgrade the general chemistry laboratory program at Georgia Southern University (GSU) through greater use of more modern laboratory techniques led to an NSF-DUE grant (0088586) to purchase computers and interfaced analytical probes. Included in the project was a complete restructuring of many of the traditional laboratory experiments to utilize the new equipment and also the development of Web-based laboratory tutorials to better prepare students for the experiments and to ease the transition into use of the new technology. Immediate improvements could be seen with the addition of computers for data acquisition, spreadsheets for data analysis, and molecular modeling software. Furthermore, the use of the Web-based tutorials served to familiarize the students with the equipment and techniques involved in the experiment, reducing the anxiety associated with using previously unseen equipment and allowing students to begin with a greater degree of confidence. This has been very important for those students with limited prior chemistry experience and was instrumental in helping students adapt to the new computer-based equipment. A particularly beneficial result of the computers and tutorials has been the savings in laboratory time, which has allowed experiments to be expanded to explore conceptual understandings and related applications.

The heme group of myeloperoxidase shows anomalous optical properties, and the enzyme possesses the unique ability to catalyze the oxidation of chloride. However, the nature of the covalently bound heme macrocycle has been difficult to identify. In this work, the electronic and magnetic properties of the heme groups in oxidized and reduced forms of wild-type and Met243Thr mutant myeloperoxidase and wild-type lactoperoxidase have been investigated using variable-temperature (1.6-273 K) magnetic circular dichroism (MCD) spectroscopy along with parallel optical absorption and electron paramagnetic resonance studies. The results provide assessment of the spin state mixtures of the oxidized and reduced samples at ambient and liquid helium temperatures and show that the anomalous MCD properties of myeloperoxidase, e.g. red-shifted and inverted signs for bands in the high-spin ferric and low-spin ferrous forms compared to other heme peroxidases and heme proteins in general, are a direct consequence of a novel electron-withdrawing sulfonium ion heme linkage involving Met243.

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