Spin-equilibrium and heme-ligand alteration in a high-potential monoheme cytochrome (cytochrome c554) from Achromobacter cycloclastes, a denitrifying organism

A c-type monoheme cytochrome c554 (13 kDa) was isolated from cells of Achromobacter cycloclastes IAM 1013 grown anaerobically as a denitrifier. The visible absorption spectrum indicates the presence of a band at 695 nm characteristic of heme-methionine coordination (low-spin form) coexisting with a minor high-spin form as revealed by the contribution at 630 nm. Magnetic susceptibility measurements support the existence of a small contribution of a high-spin form at all pH values, attaining a minimum at intermediate pH values. The mid-point redox potential determined by visible spectroscopy at pH 7.2 is +150 mV. The pH-dependent spin equilibrum and other relevant structural features were studied by 300-MHz 1H-NMR spectroscopy. In the oxidized form, the 1H-NMR spectrum shows pH dependence with pKa values at 5.0 and 8.9. According to these pKa values, three forms designated as I, II and III can be attributed to cytochrome c554. Forms I and II predominate at low pH values, and the 1H-NMR spectra reveal heme methyl proton resonances between 40 ppm and 22 ppm. These forms have a methionyl residue as a sixth ligand, and C6 methyl group of the bound methionine was identified in the low-field region of the NMR spectra. Above pH 9.6, form III predominates and the 1H-NMR spectrum is characterized by down-field hyperfine-shifted heme methyl proton resonances between 29 ppm and 22 ppm. Two new resonances are observed at congruent to 66 ppm and 54 ppm, and are taken as indicative of a new type of heme coordination (probably a lysine residue). These pH-dependent features of the 1H-NMR spectra are discussed in terms of the heme environment structure. The chemical shifts of the methyl resonances at different pH values exhibit anti-Curie temperature dependence. In the ferrous state, the 1H-NMR spectrum shows a methyl proton resonance at -3.9 ppm characteristic of methionine axial ligation. The electron-transfer rate between ferric and ferrous forms has been estimated to be smaller than 2 x 10(4) M-1 s-1 at pH 5. EPR spectroscopy was also used to probe the ferric heme environment. A prominent signal at gmax congruent to 3.58 and the overall lineshape of the spectrum indicate an almost axial heme environment.     

Hexaheme nitrite reductase from Desulfovibrio desulfuricans. M?ssbauer and EPR characterization of the heme groups

M?ssbauer and EPR spectroscopy were used to characterize the heme prosthetic groups of the nitrite reductase isolated from Desulfovibrio desulfuricans (ATCC 27774), which is a membrane-bound multiheme cytochrome capable of catalyzing the 6-electron reduction of nitrite to ammonia. At pH 7.6, the as-isolated enzyme exhibited a complex EPR spectrum consisting of a low-spin ferric heme signal at g = 2.96, 2.28, and 1.50 plus several broad resonances indicative of spin-spin interactions among the heme groups. EPR redox titration studies revealed yet another low-spin ferric heme signal at g = 3.2 and 2.14 (the third g value was undetected) and the presence of a high-spin ferric heme. M?ssbauer measurements demonstrated further that this enzyme contained six distinct heme groups: one high-spin (S = 5/2) and five low-spin (S = 1/2) ferric hemes. Characteristic hyperfine parameters for all six hemes were obtained through a detailed analysis of the M?ssbauer spectra. D. desulfuricans nitrite reductase can be reduced by chemical reductants, such as dithionite or reduced methyl viologen, or by hydrogenase under hydrogen atmosphere. Addition of nitrite to the fully reduced enzyme reoxidized all five low-spin hemes to their ferric states. The high-spin heme, however, was found to complex NO, suggesting that the high-spin heme could be the substrate binding site and that NO could be an intermediate present in an enzyme-bound form.     

Oxidation-reduction potentials of the hemes in cytochrome C3 from Desulfovibrio gigas in the presence and absence of ferredoxin by EPR spectroscopy.

1. Ferricytochrome c3 from D. gigas exhibits two low-spin ferric heme EPR resonances with gz-values at 2.959 and 2.853. Ferrocytochrome c3 is diamagnetic based on the absence of any EPR signals. 2. EPR potentiometric titrations result in the resolution of the two low-spin ferric heme resonances into two additional heme components representing in total the four hemes of the cytochrome, with EM values of -235 mV and -315 mV at heme resonance I and EM values of -235 mV and -306 mV at heme resonance II. 3. EPR spectroscopy has detected a significant diminution of intensity (approx. 60 p. 100) in the gx amplitude of ferricytochrome c3 in the presence of D. gigas ferredoxin II. The presence of ferredoxin II also causes a more negative shift in the EM of the second components of the signals at heme resonances I and II of cytochrome C3. Both observations suggest that an interaction has occurred between cytochrome C3 and ferredoxin II. 4. The results presented suggest that the heme ligand environment of ferricytochrome c3 from D. gigas is less perturbed and/or less asymmetric than environment for ferricytochrome c3 from D. vulgaris whose EPR behavior indicates the non-equivalence of all four hemes.     

Purification,characterization and biological activity of three forms of ferredoxin from the sulfate-reducing bacterium Desulfovibrio gigas

Three forms of ferredoxin FdI, FdI′, and FdII have been isolated from Desulfovibrio gigas, a sulfate reducer. They are separated by a combination of DEAE-cellulose and gel filtration chromatographic procedures. FdI and FdI′ present a slight difference in isoelectric point which enables the separation of the two forms over DEAE-cellulose, while FdII is easily separated from the two other forms by gel filtration. The three forms have the same amino acid composition and are isolated in different aggregation states. Molecular weight determinations by gel filtration gave values of 18 000 for FdI and FdI′ and 24 000 for FdII, whereas a value of 6000 is determined when dissociation is accomplished with sodium dodecyl sulfate. The electronic spectra are different and their ultraviolet-visible absorbance rations are 0.77, 0.87 and 0.68 respectively for FdI, FdI′ and FdII. Despite these differences, the physiological activities of the three forms are similar as far as the reduction of sulfite by molecular hydrogen is concerned.     

Pretreatment free of 2,4-dichlorophenoxyacetic acid improves the differentiation of sugarcane somatic embryos by affecting the hormonal balance and the accumulation of reserves

Plant Cell, Tissue and Organ Culture (PCTOC) - Several factors influence culture conditions and somatic embryogenesis responses, such as the role of plant growth regulators (PGRs) during the...