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X-ray absorption near-edge spectra (XANES) are reported for 44 Ni(I1) and Ni(II1) complexes with Nand/or S-donor ligands.The spectra reveal features associated with 1s -3d and 1s -4p, electronic transitions, whose presence or absence and intensity provide information that allows the coordination number/geometry of the complex to be determined in most cases. The complexes in this study were selected in order to examine the reliability of coordination number/geometry assignments in complexes with low symmetry and to examine the effects on the spectra of a change in formal oxidation state from +I1 to +HI. The effects on the spectra due to changes in the ligand environment are examined, and the edge energy and the breadth of the edge are found to correlate with the average hardness of the ligand environment. The effects on the spectra due to oxidation state changes are examined by using several pairs of Ni(II/III) isoleptic complexes. These compounds reveal that the effects of changes in the formal oxidation state of the Ni center are strongly dependent on the nature of the ligands present, with S-donor ligands giving rise to smaller shifts in edge energy than N,O-donor ligands. These trends are indicative of the increasing role of ligand oxidation in Ni(lI1) thiolate complexes. These trends are corroborated by X-ray photoelectron spectroscopic (XPS) studies that show a similar trend in both ligand and metal electron binding energies. The information obtained from the model studies is used to examine the Ni K-edge XANES spectrum obtained from a sample of Thiocapsa roseopersicina hydrogenase poised in form C. This spectrum is shown to be consistent with a distorted trigonal-bipyramidal geometry and a mixed 0,N-and S-donor ligand environment for this biological Ni site. ' (3) (a) Cammack, R.; Fernandez, V. M.; Schneider, K. In The Bioinorganic Chemistry of Nickel; Lancaster, J. R., Ed.; VCH: Deerfield Beach, FL, 1988; Chapter 8. (b) Moura, J. J. G.; Tiexera, M.; Moura, I.; LeGall. J. Ibid., Chapter 9. (c) Bastian, N. R.; Wink, D. A.; Wackett, L. P.; Livingston, D. J.; Jordan, L. M.; Fox, J.; Orme-Johnson, W. H.; Walsh, C. T. Ibid., Chapter 10. (4) Ragsdale, S . W.; Wood, H. G.; Morton, T. A.; Ljungdahl, L. G.; DerVartanian, D. Hasnain, S. S.; Piggott, B.; Williams, D. J. Biochem. J. 1984, 220, 591. (9) Fauque, G.; Peck, H. D., Jr.; Moura, J. J. G.; Huynh. B. H.; Berlier, Y.; DerVartanian, D. V.; Teixeira, M.; Przybyla, A. E.; Lespinat, P. A.; Moura, I.; LeGall, J. FEMS Microbiol. Rev. 1988, 54, 299. (IO) (a) Lindahl, P. A.; Kojima, N.; Hausinger, R. P.; Fox, J. A,; Tco, B. K.; Walsh, C. T.; Orme-Johnson, W. H. J. Am. Chem. Soc. 1984,106, 3062. (b) Scott, R. A.; Wallin, S . A.; Czechowski, M.; Dervartanian, D. V.; LeGall, J.; Peck, H. D., Jr.; Moura, I. J. Am. Chem. Soc. 1984, 106, 6864. (c) Scott, R. A.; Czechowski, M.; DerVartanian, D. V.; LeGall, J.; Peck, H. D., Jr.; Moura, I. Rev. Port. Quim. 1985, 27,67. (d) Albracht, S . P. J.; Kroger, A,; van der Zwaan, J. W.; Unden, G.; Bikher, R.; Mell, H.; Fontijn, R. D. Bioc...

Escherichia coli RcnR and Mycobacterium tuberculosis CsoR are the founding members of a recently identified, large family of bacterial metal-responsive DNA-binding proteins. RcnR controls the expression of the metal efflux protein RcnA only in response to Ni(II) and Co(II) ions. Here, the interaction of Ni(II) and Co(II) with wild-type and mutant RcnR proteins is examined to understand how these metals function as allosteric effectors. Both metals bind to RcnR with nanomolar affinity and stabilize the protein to denaturation. X-ray absorption and electron paramagnetic resonance spectroscopies reveal six-coordinate high-spin sites for each metal that contains a thiolate ligand. Experimental data support a tripartite N-terminal coordination motif (NH2-Xaa-NH-His) that is common for both metals. However, the Ni(II)- and Co(II)-RcnR complexes are shown to differ in the remaining coordination environment. Each metal coordinates a conserved Cys ligand but with distinct M-S distances. Co(II)-thiolate coordination has not been observed previously in Ni(II)-/Co(II)-responsive metalloregulators. The ability of RcnR to recruit ligands from the N-terminal region of the protein distinguishes it from CsoR, which uses a lower coordination geometry to bind Cu(I). These studies facilitate comparisons between Ni(II)-RcnR and NikR, the other Ni(II)-responsive transcriptional regulator in E. coli, to provide a better understanding how different nickel levels are sensed in E. coli. The characterization of the Ni(II)- and Co(II)-binding sites in RcnR, in combination with bioinformatics analysis of all RcnR/CsoR family members, identified a four amino acid fingerprint that likely defines ligand-binding specificity, leading to an emerging picture of the similarities and differences between different classes of RcnR/CsoR proteins.

Ni-containing superoxide dismutase (NiSOD) is the most recently discovered member of the class of metalloenzymes that detoxify the superoxide radical in aerobic organisms. In this study, we have employed a variety of spectroscopic and computational methods to probe the electronic structure of the NiSOD active site in both its oxidized (NiSOD(ox), possessing a low-spin (S = (1)/(2)) Ni(3+) center) and reduced (NiSOD(red), containing a diamagnetic Ni(2+) center) states. Our experimentally validated computed electronic-structure description for NiSOD(ox) reveals strong sigma-bonding interactions between Ni and the equatorial S/N ligands, which give rise to intense charge-transfer transitions in the near-UV region of the absorption spectrum. Resonance Raman (rR) spectra obtained with laser excitation in this region exhibit two features at 349 and 365 cm(-)(1) that are assigned to Ni-S(Cys) stretching modes. The NiSOD(red) active site also exhibits a high degree of metal-ligand bond covalency as well as filled/filled pi-interactions between Ni and S/N orbitals, which serve to adjust the redox potential of the Ni(2+) center. Comparison of our computational results for NiSOD(red) with those obtained in parallel studies of synthetic [NiS(2)N(2)] complexes reveals that the presence of an anionic N-donor ligand is crucial for promoting metal-based (versus S-based) oxidation of the active site. The implications of our electronic-structure descriptions with respect to the function of NiSOD are discussed, and a comparison of M-S(Cys) bonding in NiSOD and other metalloenzymes with sulfur ligation is provided.

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Superoxide dismutases are metalloenzymes involved in protecting cells from oxidative damage arising from superoxide radical or reactive oxygen species produced from superoxide. Examples of enzymes containing Cu, Mn, and Fe as the redox-active metal have been characterized. Recently, a SOD containing one Ni atom per subunit was reported. The amino acid sequence of the NiSOD deduced from the nucleotide sequence of the structural gene sodN from Streptomyces seoulensis is reported and has no homology with other SODs. X-ray absorption spectroscopic studies coupled with EPR of the Ni center show that the Ni in the oxidized (as isolated) enzyme is in a five-coordinate site composed of three S-donor ligands, one N-donor, and one other O- or N-donor. This unique coordination environment is modified by the loss of one N- (or O-) donor ligand in the dithionite-reduced enzyme. The NiSOD activity was determined by pulse radiolysis, and a value of kcat = 1.3 x 10(9) M-1 s-1 per Ni was obtained. The rate is pH sensitive and drops off rapidly above pH 8. The results characterize a novel class of metal center active in catalyzing the redox chemistry of superoxide and, when placed in context with other nickel enzymes, suggest that thiolate ligation is a prerequisite for redox-active nickel sites in metalloenzymes.

Cysteine dioxygenase (CDO) catalyzes the oxidation of cysteine to cysteine sulfinic acid, which is the first major step in cysteine catabolism in mammalian tissues. Crystal structures of mouse, rat, human and bacterial CDO have recently become available and provide significant mechanistic insights. Unlike most non-heme Fe(II) dioxygenases, coordination of the Fe in CDO deviates from the 2-His-1-carboxylate facial triad archetype and instead adopts a His3 facial triad. This change is expected to have an influence on oxygen activation by the catalytic site. The structures also reveal the presence of a cysteinyltyrosine (Tyr157-Cys93) post-translational modification near the active site. Kinetic studies of mutant CDOs reveal that the cysteine residue is less critical than the tyrosine for enzyme activity. Inconsistencies about the details of the active site and the nature of substrate binding exist and are discussed. Herein we review the structural biology along with relevant kinetics studies that have been conducted on CDO for insights into the reaction mechanism of this novel non-heme iron dioxygenase.

Studies of the transcriptional repression of the Ni-specific permease encoded by the Pnik operon by Escherichia coli NikR using a LacZ reporter assay establish that the NikR response is specific to nickel in vivo. Toward understanding this metal ion-specific response, X-ray absorption spectroscopy (XAS) analysis of various M-NikR complexes (M = Co(II), Ni(II), Cu(II), Cu(I), and Zn(II)) was used to show that each high-affinity binding site metal adopts a unique structure, with Ni(II) and Cu(II) being the only two metal ions to feature planar four-coordinate complexes. The results are consistent with an allosteric mechanism whereby the geometry and ligand selection of the metal present in the high-affinity site induce a unique conformation in NikR that subsequently influences DNA binding. The influence of the high-affinity metal on protein structure was examined using hydrogen/deuterium (H/D) exchange detected by liquid chromatography-electrospray ionization mass spectrometry (LC-ESI-MS). Each NikR complex gives rise to differing amounts of H/D exchange; Zn(II)- and Co(II)-NikR are most like apo-NikR, while the exchange time course is substantially different for Ni(II) and to a lesser extent for Cu(II). In addition to the high-affinity metal binding site, E. coli NikR has a low-affinity metal-binding site that affects DNA binding affinity. We have characterized this low-affinity site using XAS in heterobimetallic complexes of NikR. When Cu(II) occupies the high-affinity site and Ni(II) occupies the low-affinity site, the Ni K-edge XAS spectra show that the Ni site is composed of six N/O-donors. A similar low-affinity site structure is found for the NikR complex when Co(II) occupies the low-affinity site and Ni(II) occupies the high-affinity site, except that one of the Co(II) ligands is a chloride derived from the buffer.