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The nickel/iron/sulfur center of the carbon monoxide dehydrogenase (carbon monoxide:(acceptor)oxidoreductase; EC 1.2.99.2) enzyme from Rhodospirillum rubrum (Rr-CODH) was studied by x-ray absorption spectroscopy at the Ni K edge. Extended x-ray absorption fine structure data show that the first Ni coordination shell consists of 2 S atoms at 2.23 A and 2-3 N/O atoms at 1.87 A. The edge structure indicates a distorted tetrahedral or five-coordinate Ni environment in both oxidized and reduced Rr-CODH. By comparing second-shell extended x-ray absorption fine structure data of Rr-CODH to that of (Et4N)3[NiFe3S4(SEt)4], a cubane-type cluster, it was clearly established that Ni in the Rr-CODH center is not involved in the core of a NiFe3S4 cubane cluster. One model consistent with the results is a mononuclear The metabolic pathways of certain bacteria involve conversion of CO to CO2 catalyzed by a Ni-containing enzyme, carbon monoxide dehydrogenase [CODH; carbon-monoxide: (acceptor)oxidoreductase; EC 1.2.99.2], now purified from several anaerobes (1-12). Some of these enzymes effect the synthesis of acetyl coenzyme A by means of the Wood pathway (13,14). Recent extended x-ray absorption fine structure (EXAFS) and x-ray absorption spectroscopy (XAS) edge studies of the CO-free, electron paramagnetic resonance-silent form of CODH from Clostridium thermoaceticum indicate a NiS4 (15) or =NiS2(O/N)2 (16) coordination environment with distorted square-planar or pyramidal geometry (16). EXAFS further suggests that an Fe-S cluster may be nearby (15). The CODH from Rhodospirillum rubrum (Rr-CODH) is a simpler enzyme (62-kDa monomer, 1 Ni and 8 Fe/S) that only oxidizes CO (8, 9). Isotopically induced electron paramagnetic resonance line-broadening suggests that Ni is covalently coupled to one or both Fe4S4 clusters in Rr-CODH and in other enzymes as well (17)(18)(19)(20). The possibility of an NiFe3S4 cubane-type cluster has been recognized (20), and alternative models exist in which Ni is bridged to Fe-S centers. Elucidation of the Ni-site structure is essential to understanding its role in catalysis. Toward that end, we report Ni K-edge XAS results for Rr-CODH. MATERIALS AND METHODSSample Preparations. Rr-CODH was purified and assayed as described (21). Purified Rr-CODH had a specific activity of 5200 gmol of CO oxidized per min-mg and contained 1.2 Ni and 8.1 Fe per mol of enzyme as determined by plasma emission spectroscopy. Rr-CODH samples were prepared for spectroscopic studies in an anaerobic glove box [Vacuum Atmospheres (Hawthorne, CA) Dri-Lab glove box model HE-493] containing an N2 atmosphere with <1 ppm 02. Rr-CODH samples in 100 mM Mops buffer, pH 7.5, were oxidized with indigo carmine as described (22) and were concentrated to 1.25 mM in a collodion ultrafiltration apparatus (Schleicher & Schuell). Some of the concentrated protein sample was reduced by adding sodium dithionite (5 mM final concentration dithionite). The oxidized and reduced samples were loaded into "140 IL XAS lucite cells (23 x 2 x 3 mm) wi...

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X-ray absorption spectroscopy (XAS) is a useful tool for obtaining structural and chemical information about the active sites of metalloproteins and metalloenzymes. Information may be obtained from both the edge region and the extended X-ray absorption fine structure (EXAFS) or post-edge region of the K-edge X-ray absorption spectrum of a met_ center in a compound. The edge contains information about the valence electronic structure of the atom that absorbs the X-rays. lt is possible in some systems to infer the redox state of the metal atom in question, as well as the geometry and nature of ligands connected to it, from the features in the edge in a straightforward manner. The EXAFS modulationsl being produced by the backscattering of the ejected photoelectron from the atoms surrounding the metal atom, provide, when analyzed, information about the number and type of neighbouring atoms, and the distances at which they occur. In this thesis, analysis of both the edge and EXAFS regions has been used to gain information about the active sites of various metalloproteins. The metalloproteins studied were plastocyanin (Pc), laccase and nickel carbon monoxide dehydrogenase (Ni CODH). Studies of Cu(I)-imidazole compounds, related to the protein hemocyanin, are also reported here. Hemocyanin (Hc) is an oxygen transport protein containing two copper atoms in its active site. In oxy Hc (when dioxygen is bound), the EXAFS interaction between the two Cu atoms is readily seen, but in deoxy Hc (no 02 is bound and both Cu ions are in the Cu(I) state) the Cu-Cu interaction is weak and difficult to distinguish from the strong EXAFS signal produced by imidazole rings (from histidine) ligated to both Cu. Some studies of Cu(I)-imidazole compounds were thus made, with a view towards accurately interpreting the EXAFS spectrum of hemocyanin in the reduced or deoxy state. The crystal structure of Cu(I)(N-methylimidazole)2BF4 was solved and is reported here. We also made an attempt to better characterize the EXAFS of Cu-imidazole by relating the EXAFS Debye-Waller factor from spectra of Cu(I)(N-methylimidazole)4CIO4 measured over 10-200 K to the vibrational frequencies of the Cu-imidazole interactions. We found that the EXAFS Debye-Waller factor is not determined wit!a sufficient accuracy for us to solve an EXAFS temperature series for a unique value of a bend-stretch frequency or force constant. Plastocyanin is a small (99-amino acid residue) protein, containing one copper atom, that functions as a electron carrier in the process of photosynthesis. This protein, along with other "blue copper" proteins, is of special interest to inorganic chemists because of its unique spectroscopic features, including an unusually intense blue colour, which arise from