Formation of a stable cyano-bridged dinuclear iron cluster following oxidation of the superoxide reductases from Treponema pallidum and Desulfovibrio vulgaris with K(3)Fe(CN)(6)

Inorg. Chem., 2003, 42 (4), pp 938–940 DOI: 10.1021/ic0262886Superoxide reductases catalyze the monovalent reduction of superoxide anion to hydrogen peroxide. Spectroscopic evidence for the formation of a dinuclear cyano-bridged adduct after K(3)Fe(CN)(6) oxidation of the superoxide reductases neelaredoxin from Treponema pallidum and desulfoferrodoxin from Desulfovibrio vulgaris was reported. Oxidation with K(3)Fe(CN)(6) reveals a band in the near-IR with lambda(max) at 1020 nm, coupled with an increase of the iron content by almost 2-fold. Fourier transform infrared spectroscopy provided additional evidence with CN-stretching vibrations at 2095, 2025-2030, and 2047 cm(-)(1), assigned to a ferrocyanide adduct of the enzyme. Interestingly, the low-temperature electronic paramagnetic resonance (EPR) spectra of oxidized TpNlr reveal at least three different species indicating structural heterogeneity in the coordination environment of the active site Fe ion. Given the likely 6-coordinate geometry of the active site Fe(3+) ion in the ferrocyanide adduct, we propose that the rhombic EPR species can serve as a model of a hexacoordinate form of the active site

Resolving subunit degeneracy with nonsymmetric pseudocontact shifts

Protein Science (2002), 11:2464–2470Desulfovibrio gigas desulforedoxin (Dx) consists of two identical peptides, each containing one [Fe-4S]center per monomer. Variants with different iron and zinc metal compositions arise when desulforedoxin is produced recombinantly from Escherichia coli. The three forms of the protein, the two homodimers [Fe(III)/Fe(III)]Dx and [Zn(II)/Zn(II)]Dx, and the heterodimer [Fe(III)/Zn(II)]Dx, can be separated by ion exchange chromatography on the basis of their charge differences. Once separated, the desulforedoxins containing iron can be reduced with added dithionite. For NMR studies, different protein samples were prepared labeled with 15N or 15N + 13C. Spectral assignments were determined for [Fe(II)/Fe(II)]Dx and [Fe(II)/Zn(II)]Dx from 3D 15N TOCSY-HSQC and NOESY-HSQC data, and compared with those reported previously for [Zn(II)/Zn(II)]Dx. Assignments for the 13C shifts were obtained from an HNCA experiment. Comparison of 1H–15N HSQC spectra of [Zn(II)/Zn(II)]Dx, [Fe(II)/Fe(II)]Dx and [Fe(II)/Zn(II)]Dx revealed that the pseudocontact shifts in [Fe(II)/Zn(II)]Dx can be decomposed into inter- and intramonomer components, which, when summed, accurately predict the observed pseudocontact shifts observed for [Fe(II)/Fe(II)]Dx. The degree of linearity observed in the pseudocontact shifts for residues 8.5 Å from the metal center indicates that the replacement of Fe(II) by Zn(II) produces little or no change in the structure of Dx. The results suggest a general strategy for the analysis of NMR spectra of homo-oligomeric proteins in which a paramagnetic center introduced into a single subunit is used to break the magnetic symmetry and make it possible to obtain distance constraints (both pseudocontact and NOE) between subunits

Requirements for Nitric Oxide Generation from Isoniazid Activation In Vitro and Inhibition of Mycobacterial Respiration In Vivo

Isoniazid (INH), a front-line antituberculosis agent, is activated by mycobacterial catalase-peroxidase KatG, converting INH into bactericidal reactive species. Here we investigated the requirements and the pathway of nitric oxide (NO ˙) generation during oxidative activation of INH by Mycobacterium tuberculosis KatG in vitro. We also provide in vivo evidence that INH-derived NO ˙ can inhibit key mycobacterial respiratory enzymes, which may contribute to the overall antimycobacterial action of INH