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Rubredoxins (Rds) and their derivatives have been extensively used, in the last few decades, in order to elucidate structure and functional aspects of metal sites involving rich sulphur coordination spheres. The derivatives have been designed with two main purposes: (a) replacement of selective and specific amino acid residues in native systems by site direct mutagenesis, and (b) replacement of native metal ion (iron), producing novel metal sites. We will highlight in this review the key amino acid residues, recognized in native Rds, directly involved in electron transfer mechanisms. By protein template assisted synthesis, metal-substituted Rds are used as structural probes and bio-models. The tetra-cysteinyl metal coordination site in Rd has the surprising capacity of chelating a wide variety of metal ions (other than native iron), and this chemistry has been witnessing significant growth based on two main interests: (i) metals such as 57 Fe, Zn, Co, Cd, Ga, In, and Hg were introduced and derivatives were synthesized in order to specifically probe structural determinants imposed by the protein, and (ii) Zn, Ni, Cu, as well as Mo (and most recently, W), were used with a particular interest: to model complex metal centres in sulphur-rich enzymes. The latter, in particular, is discussed in the context of Rd serving as a model complex of native enzymes, as well as the synthesis of small inorganic complexes. The review aims to bring synthetic biochemistry and synthetic inorganic chemistry together in a synergistic way. [ABSTRACT FROM AUTHOR]

Abstract: Cytochrome c nitrite reductase is a multicenter enzyme that uses a five-coordinated heme to perform the six-electron reduction of nitrite to ammonium. In the sulfate reducing bacterium Desulfovibrio desulfuricans ATCC 27774, the enzyme is purified as a NrfA2NrfH complex that houses 14 hemes. The number of closely-spaced hemes in this enzyme and the magnetic interactions between them make it very difficult to study the active site by using traditional spectroscopic approaches such as EPR or UV–Vis. Here, we use both catalytic and non-catalytic protein film voltammetry to simply and unambiguously determine the reduction potential of the catalytic heme over a wide range of pH and we demonstrate that proton transfer is coupled to electron transfer at the active site. [Copyright &y& Elsevier]

In this work we present a kinetic study of the superoxide-mediated electron transfer reactions between rubredoxin-type proteins and members of the three different classes of superoxide reductases (SORs). SORs from the sulfate-reducing bacteria Desulfovibrio vulgaris ( Dv) and D. gigas ( Dg) were chosen as prototypes of classes I and II, respectively, while SOR from the syphilis spyrochete Treponema pallidum ( Tp) was representative of class III. Our results show evidence for different behaviors of SORs toward electron acceptance, with a trend to specificity for the electron donor and acceptor from the same organism. Comparison of the different k app values, 176.9±25.0 min−1 in the case of the Tp/ Tp electron transfer, 31.8±3.6 min−1 for the Dg/ Dg electron transfer, and 6.9±1.3 min−1 for Dv/ Dv, could suggest an adaptation of the superoxide-mediated electron transfer efficiency to various environmental conditions. We also demonstrate that, in Dg, another iron–sulfur protein, a desulforedoxin, is able to transfer electrons to SOR more efficiently than rubredoxin, with a k app value of 108.8±12.0 min−1, and was then assigned as the potential physiological electron donor in this organism. [ABSTRACT FROM AUTHOR]

Superoxide reductases are a class of non-haem iron enzymes which catalyse the monovalent reduction of the superoxide anioninto hydrogen peroxide and water.Treponema pallidum(Tp), the syphilis spirochete, expresses the gene for a superoxide reductase called neelaredoxin, having the iron protein rubredoxin as the putative electron donor necessary to complete the catalytic cycle. In this work, we present the first cloning, overexpression inEscherichia coliand purification of the Tp rubredoxin. Spectroscopic characterization of this 6 kDa protein allowed us to calculate the molar absorption coefficient of the 490 nm feature of ferric iron, e=6.9±0.4 mM-1 cm-1. Moreover, the midpoint potential of Tp rubredoxin, determined using a glassy carbon electrode, was -76±5 mV. Reduced rubredoxin can be efficiently reoxidized upon addition of Na2IrCl6-oxidized neelaredoxin, in agreement with a direct electron transfer between the two proteins, with a stoichiometry of the electron transfer reaction of one molecule of oxidized rubredoxin per one molecule of neelaredoxin. In addition, in presence of a steady-state concentration of superoxide anion, the physiological substrate of neelaredoxin, reoxidation of rubredoxin was also observed in presence of catalytic amounts of superoxide reductase, and the rate of rubredoxin reoxidation was shown to be proportional to the concentration of neelaredoxin, in agreement with a bimolecular reaction, with a calculatedkapp=180 min-1. Interestingly, similar experiments performed with a rubredoxin from the sulfate-reducing bacteriaDesulfovibrio vulgarisresulted in a much lower value ofkapp=4.5 min-1. Altogether, these results demonstrated the existence for a superoxide-mediated electron transfer between rubredoxin and neelaredoxin and confirmed the physiological character of this electron transfer reaction. [ABSTRACT FROM AUTHOR]