. DpnM, and by extension the DpnM family or group alpha Mtases, contains the consensus fold and AdoMet-binding motifs found in most Mtases. Structural considerations suggest that macromolecular Mtases evolved from small-molecule Mtases, with different groups of DNA Mtases evolving independently. Mtases may have evolved from dehydrogenases. Comparison of these enzymes indicates that in protein evolution, the structural fold is most highly conserved, then function and lastly sequence.

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...

An EXAFS study on the oxidized and reduced forms of cytochromes c' from Rhodospirillum rubrum and Rhodospirillum molischianum was performed at pH 7. The cytochromes c' have an apparent coordination number of 5 in both oxidation states. Average Fe-ligand bond lengths of 2.02 +/- 0.025 and 2.06 +/- 0.025 A are obtained in their oxidized and reduced forms, respectively. By use of suitable values for the Fe-NHis bond length and Fe out-of-plane displacement, as determined by small molecule crystallographic techniques, the Fe-Npyrrole bond lengths and the porphyrin center-to-Npyrrole distance have been estimated for cytochrome c' in both of its oxidation states. With this model, estimates of the Fe-Npyrrole bond lengths are 2.01 +/- 0.03 and 2.05 +/- 0.03 A, for the oxidized and reduced cytochromes c', respectively. The center-to-Npyrrole distance is estimated to be 1.99 +/- 0.03 A for oxidized cytochrome c' and 2.03 +/- 0.03 A for reduced cytochrome c'.

EXAFS fluorescence spectra were recorded for high-potential c-type cytochromes which range in oxidation-reduction potential from +145 to +365 mV. No average Fe-ligand bond length differences greater than 0.03 A were observed, for any cytochrome source of oxidation state. Least-squares analysis in combination with model calculations allowed limits to be set on the average Fe-N bond length (1.97-1.99 A) and the Fe-S bond length (2.29-2.32 A). A change of 0.05 A in either the average Fe-N or the Fe-S bond length is readily detectable with the range and quality of the data presented here. Two major conclusions are drawn from this study: In octahedrally coordinated iron porphyrin systems, Fe-N and Fe-S bond lengths are independent of oxidation-reduction potential, and they are also independent of oxidation state. A model for the regulation of oxidation-reduction potential in cytochrome c is proposed.

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