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Copper is an essential element that becomes highly cytotoxic when concentrations exceed the capacity of cells to sequester the ion. Here, we identify a new copper-specific repressor (CsoR) of a copper-sensitive operon (cso) in Mycobacterium tuberculosis (Mtb) that is representative of a large, previously uncharacterized family of proteins (DUF156). Electronic and X-ray absorption spectroscopies reveal that CsoR binds a single-monomer mole equivalent of Cu(I) to form a trigonally coordinated (S(2)N) Cu(I) complex. The 2.6-A crystal structure of copper-loaded CsoR shows a homodimeric antiparallel four-helix bundle architecture that represents a novel DNA-binding fold. The Cu(I) is coordinated by Cys36, Cys65' and His61' in a subunit bridging site. Cu(I) binding negatively regulates the binding of CsoR to a DNA fragment encompassing the operator-promoter region of the Mtb cso operon; this results in derepression of the operon in Mtb and the heterologous host Mycobacterium smegmatis. Substitution of Cys36 or His61 with alanine abolishes Cu(I)- and CsoR-dependent regulation in vivo and in vitro. Potential roles of CsoR in Mtb pathogenesis are discussed.

SQUID Magnetic Measurements on#1 -z+1,x-1/2,-y+1/2 Positional parameters and B(eq) for 2.62(5)2.40(5) 2.1(4) 2.4(4) 2.4(4) 2.3(4) 2.0(4) 2.0(4) 2.1(5) 2.7(6) 2.5(5) 3.2(6) 3.0(6) 3.9(7) 3.1(6) 4.3(7) 2.5(5) 3.7(6) 2.8(6) 3.1(6) 3.8(6) 3.3(6) 2.2(5) 2.2(5) 3.0(6) 2.6(5) 2.9(6) 3.5(6) 3.1(6) 3.7(7) 2.8(6) 3.3(6) 3.0(6) 2.5(5) 2.8(6) 3.0(6) 2.4(5) 2.6(5) 2.9(6) 2.5(5) 3.8(7) 3.3(6) 4.0(7) 3.7(7) 3.2(6) 3.2(6) 2.4(5) 2.9(6) 2.6(5) 0 1996 American Chemical Society J. Am. Chem. Soc. VI 18 Page8623 Laplaza Supplemental Page 9Positional parameters and B(eq) for 0.044(7) 0.038 (7 1.960 (7) 1.964 (7) 1.977 (7) 1.970 (7) 1.968 (7) 1.956 (7) 1.42 (1) 1.50(1)1.42 (1) 1.48 (1) 1.44 (1) 1.49 ( 122.8(9)122 (1) 117 ( 120 (1) 120 (1) 120 (

The cytosolic C-terminal domain of the membrane copper transporter Ctr1 from the yeast Saccharomyces cerevisiae, Ctr1c, was expressed in E. coli as an oxygen-sensitive soluble protein with no significant secondary structure. Visible-UV spectroscopy demonstrated that Ctr1c bound four Cu(I) ions, structurally identified as a Cu(I)(4)(micro-S-Cys)(6) cluster by Xray absorption spectroscopy. This was the only metalated form detected by electrospray ionization mass spectrometry. An average dissociation constant K(D) = (K(1)K(2)K(3)K(4))(1/4) = 10(-)(19) for binding of Cu(I) to Ctr1c was estimated via competition with the ligand bathocuproine disulfonate bcs (beta(2) = 10(19.8)). Equivalent experiments for the yeast chaperone Atx1 and an N-terminal domain of the yeast Golgi pump Ccc2, which both bind a single Cu(I) ion, provided similar K(D) values. The estimates of K(D) were supported by independent estimates of the equilibrium constants K(ex) for exchange of Cu(I) between pairs of these three proteins. It is apparent that, in vitro, the three proteins buffer "free" Cu(I) concentrations in a narrow range around 10(-)(19) M. The results provide quantitative support for the proposals that, in yeast, (a) "free" copper concentrations are very low in the cytosol and (b) the Cu(I) trafficking gradient is shallow along the putative Ctrlc --> Atx1 --> Ccc2n metabolic pathway. In addition, both Ctr1c and its copper-responsive transcription factor Mac1 contain similar clusters which may be important in signaling copper status in yeast.

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Molybdenum K-edge X-ray absorption and Mo(V) electron paramagentic resonance (EPR) spectroscopies have been used to probe the metal coordination in oxidized and reduced forms of both wild-type and a site-directed mutant of Rhodobacter sphaeroides dimethyl sulfoxide (DMSO) reductase. We confirm our earlier findings (George, G. N.; Hilton, J.; Rajagopalan, K. V. J. Am. Chem. Soc. 1996, 118, 1113−1117) that the molybdenum site of the oxidized Mo(VI) enzyme possesses one terminal oxygen ligand (MoO) at 1.68 Å, four thiolate ligands at 2.44 Å, and one oxygen at 1.92 Å and that the dithionite-reduced Mo(IV) enzyme possesses a desoxo species with three or four Mo−S at 2.33 Å and two different Mo−O ligands at 2.16 and 1.92 Å. Mo(V) EPR indicates the presence of one exchangeable oxygen ligand, most likely an Mo−OH, in the signal-giving species, probably originating from the MoO of the oxidized enzyme (E m8.5(IV/V) = +37 mV, E m8.5(V/VI) = +83 mV). The addition of dimethyl sulfide, in the reverse of the physiological reaction, reduces the enzyme. In this form, the enzyme contains a desoxo active site with four Mo−S at 2.36 Å and two different Mo−O ligands at 1.94 and 2.14 Å. Recombinant wild-type R. sphaeroides DMSO reductase expressed in Escherichia coli initially has a dioxo structure (two MoO at 1.72 Å and four Mo−S at 2.48 Å) but assumes the wild-type Mo(VI) structure after a cycle of reduction and reoxidation. The site-directed Ser147→Cys mutant possesses a monooxo active site in the oxidized state (MoO at 1.70 Å) with five sulfur ligands (at 2.40 Å), consistent with cysteine 147 coordination to Mo. The dithionite reduced form of the mutant possesses a desoxo site also with five Mo−S ligands (at 2.37 Å) and one Mo−O at 2.12 Å. The variant has substantially different Mo(V) EPR and electrochemistry (E m8.5(IV/V) = −43 mV, E m8.5(V/VI) = +106 mV). The active-site structure and catalytic mechanism of DMSO reductase are discussed in the light of these results.

The combination of UV/visible/NIR absorption, CD and variable-temperature magnetic circular dichroism (VTMCD), EPR, and X-ray absorption (XAS) spectroscopies has been used to investigate the electronic and structural properties of the oxidized and reduced forms of Pyrococcus furiosus superoxide reductase (SOR) as a function of pH and exogenous ligand binding. XAS shows that the mononuclear ferric center in the oxidized enzyme is very susceptible to photoreduction in the X-ray beam. This observation facilitates interpretation of ground- and excited-state electronic properties and the EXAFS results for the oxidized enzyme in terms of the published X-ray crystallographic data (Yeh, A. P.; Hu, Y.; Jenney, F. E.; Adams, M. W. W.; Rees, D. C. Biochemistry 2000, 39, 2499-2508). In the oxidized state, the mononuclear ferric active site has octahedral coordination with four equatorial histidyl ligands and axial cysteinate and monodentate glutamate ligands. Fe EXAFS are best fit by one Fe-S at 2.36 A and five Fe-N/O at an average distance of 2.12 A. The EPR-determined spin Hamiltonian parameters for the high-spin (S = (5)/(2)) ferric site in the resting enzyme, D = -0.50 +/- 0.05 cm(-1) and E/D = 0.06, are consistent with tetragonally compressed octahedral coordination geometry. UV/visible absorption and VTMCD studies facilitate resolution and assignment of pi His --> Fe(3+)(t(2g)) and (Cys)S(p) --> Fe(3+)(t(2g)) charge-transfer transitions, and the polarizations deduced from MCD saturation magnetization studies indicate that the zero-field splitting (compression) axis corresponds to one of the axes with trans-histidyl ligands. EPR and VTMCD studies provide evidence of azide, ferrocyanide, hydroxide, and cyanide binding via displacement of the glutamate ligand. For azide, ferrocyanide, and hydroxide, ligand binding occurs with retention of the high-spin (S = 5/2) ground state (E/D = 0.27 and D < 0 for azide and ferrocyanide; E/D = 0.25 and D = +1.1 +/- 0.2 cm(-1) for hydroxide), whereas cyanide binding results in a low-spin (S = 1/2) species (g = 2.29, 2.25, 1.94). The ground-state and charge-transfer/ligand-field excited-state properties of the low-spin cyanide-bound derivative are shown to be consistent with a tetragonally elongated octahedral coordination with the elongation axis corresponding to an axis with trans-histidyl ligands. In the reduced state, the ferrous site of SOR is shown to have square-pyramidal coordination geometry in frozen solution with four equatorial histidines and one axial cysteine on the basis of XAS and UV and NIR VTMCD studies. Fe EXAFS are best fit by one Fe-S at 2.37 A and four Fe-N/O at an average distance of 2.15 A. VTMCD reveals a high-spin (S = 2) ferrous site with (Cys)S(p) --> Fe(2+) charge-transfer transitions in the UV region and (5)T(2g) --> (5)E(g) ligand-field transitions in the NIR region at 12400 and <5000 cm(-1). The ligand-field bands indicate square-pyramidal coordination geometry with 10Dq < 8700 cm(-1) and a large excited-state splitting, Delta (5)E(g) > 7400 cm(-1). Analys...

The fern Pteris vittata accumulates unusually high levels of arsenic. Using X-ray absorption spectroscopy (XAS) and XAS imaging, we reveal the distribution of arsenic species in vivo. Arsenate is transported through the vascular tissue from the roots to the fronds (leaves), where it is reduced to arsenite and stored at high concentrations. Arsenic-thiolate species surrounding veins may be intermediates in this reduction. In gametophytes, arsenite is compartmentalized within the cell vacuole. Arsenic is excluded from cell walls, rhizoids, and reproductive areas. This study provides important insights into arsenic hyperaccumulation, which may prove useful for phytoremediating arsenic-contaminated sites, and demonstrates the strengths of XAS imaging for distinguishing highly localized species.