Amino-acid sequence of rusticyanin from Thiobacillus ferrooxidans and its comparison with other blue copper proteins.

Rusticyanin, a copper protein characterized by a high redox potential (+680 mV) and a high stability at acidic pH, is involved in iron oxidation in Thiobacillus ferrooxidans. It has been characterized from a new strain and its amino-acid sequence has been determined and compared to two other rusticyanin sequences isolated from different strains. It comprises 155 amino acids and the alignment of the three rusticyanins shows a high degree of homology. Comparing the rusticyanins with six blue copper proteins which have a copper-I site in common, a consensus sequence containing Cys, His and Met in the C-terminal part of the protein and His-85 is proposed to be involved in the copper coordination. Secondary structure predictions are compared to three structures of copper proteins obtained by X-ray crystallography.

Active site geometry in the high oxido-reduction potential rusticyanin from Thiobacillus ferrooxidans.

Rusticyanin is a blue copper protein involved in the oxidation of iron catalyzed by Thiobacillus ferrooxidans. This protein is characterized by a high oxido-reduction potential and a high stability at low pH. The three dimensional structure of this protein is still unknown and in order to investigate the geometric properties of the copper center which could be correlated to the high oxido-reduction potential, we have studied rusticyanin by UV-Visible, EPR and NMR spectroscopies, at different pH values. Our results suggest that rusticyanin is stable between pH 2 and pH 9 and that the copper center does not undergo significant geometric modifications in this pH range. Moreover, the copper atom could be buried more deeply in the protein than in other type I copper proteins and the atomic distance Cu-S(Met), one of the four bonds involved in copper coordination, is probably shorter in rusticyanin than in other cupredoxins. These two properties of the copper site are expected to be responsible, in part, for the high oxido-reduction potential observed in rusticyanin.

Biotin synthase, a new member of the family of enzymes which uses S-adenosylmethionine as a source of deoxyadenosyl radical.

The fact that biotin synthase, from Escherichia coli and Bacillus sphaericus, requires S-adenosylmethionine and a reducing system led us to postulate that this synthase could belong to the family of enzymes which use S-adenosylmethionine as a source of deoxyadenosyl radical, namely pyruvate formate-lyase, lysine 2,3-aminomutase, and anaerobic ribonucleotide reductase. We describe here experiments with S-[2,8-(3)H] adenosylmethionine and S-adenosyl-[methyl-3H]methionine which allowed the identification and quantification of the expected cleavage products, deoxyadenosine, and methionine. They are formed in equimolar amounts, in a ratio close to 3 with respect to the biotin produced. We postulate a mechanism involving the homolytic cleavage of two C-H bonds which should consume two equivalents of S-adenosylmethionine. The observed excess of S-adenosylmethionine consumption is attributed to abortive processes.

Formation of active heterologous nitrate reductases between nitrate reductases A and Z of Escherichia coli.

Two nitrate reductases, NRA and NRZ, are present in Escherichia coli. These isoenzymes have the same alpha beta gamma, subunits composition and have similar size and genetic organization. Corresponding subunits of the complexes share at least 75% identity. By subcloning the different genes and expressing them from separate transcriptional units, we have demonstrated (i) that the translation of the subunits and their assembly are not coupled processes, since subunits produced concomitantly but independently can meet efficiently and associate to form active enzymes, and (ii) that the alpha subunit of a given complex can be replaced by its counterpart from the other isoenzyme to yield an active membrane-bound heterologous enzyme. One such heterologous enzyme, alpha A beta Z gamma Z, has been purified; it is less stable than the native enzymes, more susceptible to thermal denaturation, and shows increased sensitivity to proteolysis. It is also less stably bound to the membrane and, consequently, its activity with physiological electron donors is drastically reduced. The possibility that heterologous nitrate reductases could be formed in vivo is discussed with reference to the existence of porin heterotrimers of the outer membrane proteins OmpC, OmpF and PhoE.