M. Bruschi - Academia.edu (original) (raw)

Papers by M. Bruschi

Research paper thumbnail of Biochemical and spectroscopic characterization of two new cytochromes isolated from Desulfuromonas acetoxidans

The mitochondrial amidoxime reducing component mARC is a newly discovered molybdenum enzyme that ... more The mitochondrial amidoxime reducing component mARC is a newly discovered molybdenum enzyme that is presumed to form the catalytical part of a three-component enzyme system, consisting of mARC, heme/cytochrome b 5 , and NADH/FADdependent cytochrome b 5 reductase. mARC proteins share a significant degree of homology to the molybdenum cofactorbinding domain of eukaryotic molybdenum cofactor sulfurase proteins, the latter catalyzing the post-translational activation of aldehyde oxidase and xanthine oxidoreductase. The human genome harbors two mARC genes, referred to as hmARC-1/ MOSC-1 and hmARC-2/MOSC-2, which are organized in a tandem arrangement on chromosome 1. Recombinant expression of hmARC-1 and hmARC-2 proteins in Escherichia coli reveals that both proteins are monomeric in their active forms, which is in contrast to all other eukaryotic molybdenum enzymes that act as homo-or heterodimers. Both hmARC-1 and hmARC-2 catalyze the N-reduction of a variety of N-hydroxylated substrates such as N-hydroxy-cytosine, albeit with different specificities. Reconstitution of active molybdenum cofactor onto recombinant hmARC-1 and hmARC-2 proteins in the absence of sulfur indicates that mARC proteins do not belong to the xanthine oxidase family of molybdenum enzymes. Moreover, they also appear to be different from the sulfite oxidase family, because no cysteine residue could be identified as a putative ligand of the molybdenum atom. This suggests that the hmARC proteins and sulfurase represent members of a new family of molybdenum enzymes. . 2 The abbreviations used are: Moco, molybdenum cofactor; AO, aldehyde oxidase; cyt b 5 , cytochrome b 5 ; cyt b 5 R, cyt b 5 reductase; MCP, Moco carrier protein; MPT, molybdopterin; NR, nitrate reductase; SO, sulfite oxidase; TCEP, Tris(2-carboxyethyl) phosphine hydrochloride; XOR, xanthine oxidoreductase; XO, xanthine oxidase; Mo-enzyme, Moco-containing enzyme.

Research paper thumbnail of Some comparisons between two crystallized anaerobic bacterial rubredoxins from Desulfovibrio gigas and D. vulgaris

Journal of Molecular Biology, 1976

ABSTRACT

Research paper thumbnail of Kinetic studies on the electron transfer between bacterial c-type cytochromes and metal oxides

Journal of Electroanalytical Chemistry, 1998

Cyclic voltammetry was used to investigate the kinetics of the electron transfer between various ... more Cyclic voltammetry was used to investigate the kinetics of the electron transfer between various soluble or solid metal oxides, and polyheme c-type cytochromes from Desulfuromonas acetoxidans and Desulfo6ibrio. The second order rate constant for the catalytic reduction of soluble chromate ions by Desulfuromonas acetoxidans cytochrome c 7 was found to be 6 × 10 5 M − 1 s − 1 . By using the membrane electrode technology, it has been shown that the catalytic process for Cr(VI) reduction is efficient even when the cytochrome is entrapped in the close vicinity of the electrode surface. Moreover, this proceeding allowed the catalytic reduction of solid metal oxides such as manganese(IV), vanadium(V) and iron(III) oxides to be performed. Results suggest that the metal reductase activity of a microorganism is governed by its c-type cytochrome content. Furthermore, only cytochromes with bishistidinyl heme iron coordination act as metal reducers whereas mitochondrial c-type cytochromes do not. This approach opens new pathways for the use of sulfur or sulfate bacteria in the bioremediation of metal contaminated waters and waste streams. Processes involving the use of entrapped enzymes reactors could be developed according to the metal reducing activity of their polyheme c-type cytochromes.

Research paper thumbnail of The amino acid sequence of desulforedoxin, a new type of non heme iron protein from

Biochemical and Biophysical Research Communications, 1979

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

Biochimica et Biophysica Acta (BBA) - Protein Structure and Molecular Enzymology, 1993

Rusticyanin, a copper protein characterized by a high redox potential (+680 mV) and a high stabil... more 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.

Research paper thumbnail of Insight into Molecular Stability and Physiological Properties of the Diheme Cytochrome CYC 41 from the Acidophilic Bacterium Acidithiobacillus ferrooxidans

Biochemistry, 2005

The cyc1 gene encoding the soluble dihemic cytochrome c CYC(41) from Acidithiobacillus ferrooxida... more The cyc1 gene encoding the soluble dihemic cytochrome c CYC(41) from Acidithiobacillus ferrooxidans, an acidophilic organism, has been cloned and expressed in Escherichia coli as the host organism. The cytochrome was successfully produced and folded only in fermentative conditions: this allowed us to determine the molecular basis of the heme insertion at extreme pH. Point mutations at two sequence positions (E121 and Y63) were introduced near the two hemes in order to assign individual redox potentials to the hemes and to identify the interaction sites with the redox partners, rusticyanin and cytochrome oxidase. Characterization of mutants E121A, Y63A, and Y63F CYC(41) with biochemical and biophysical techniques were carried out. Substitution of tyrosine 63 by phenylalanine alters the environment of heme B. This result indicates that heme B has the lower redox potential. Interaction studies with the two physiological partners indicate that CYC(41) functions as an electron wire between RCy and cytochrome oxidase. A specific glutamate residue (E121) located near heme A is directly involved in the interaction with RCy. A docking analysis of CYC(41), RCy, and cytochrome oxidase allowed us to propose a model for the complex in agreement with our experimental data.

Research paper thumbnail of Biochemical and spectroscopic characterization of two new cytochromes isolated from Desulfuromonas acetoxidans

The mitochondrial amidoxime reducing component mARC is a newly discovered molybdenum enzyme that ... more The mitochondrial amidoxime reducing component mARC is a newly discovered molybdenum enzyme that is presumed to form the catalytical part of a three-component enzyme system, consisting of mARC, heme/cytochrome b 5 , and NADH/FADdependent cytochrome b 5 reductase. mARC proteins share a significant degree of homology to the molybdenum cofactorbinding domain of eukaryotic molybdenum cofactor sulfurase proteins, the latter catalyzing the post-translational activation of aldehyde oxidase and xanthine oxidoreductase. The human genome harbors two mARC genes, referred to as hmARC-1/ MOSC-1 and hmARC-2/MOSC-2, which are organized in a tandem arrangement on chromosome 1. Recombinant expression of hmARC-1 and hmARC-2 proteins in Escherichia coli reveals that both proteins are monomeric in their active forms, which is in contrast to all other eukaryotic molybdenum enzymes that act as homo-or heterodimers. Both hmARC-1 and hmARC-2 catalyze the N-reduction of a variety of N-hydroxylated substrates such as N-hydroxy-cytosine, albeit with different specificities. Reconstitution of active molybdenum cofactor onto recombinant hmARC-1 and hmARC-2 proteins in the absence of sulfur indicates that mARC proteins do not belong to the xanthine oxidase family of molybdenum enzymes. Moreover, they also appear to be different from the sulfite oxidase family, because no cysteine residue could be identified as a putative ligand of the molybdenum atom. This suggests that the hmARC proteins and sulfurase represent members of a new family of molybdenum enzymes. . 2 The abbreviations used are: Moco, molybdenum cofactor; AO, aldehyde oxidase; cyt b 5 , cytochrome b 5 ; cyt b 5 R, cyt b 5 reductase; MCP, Moco carrier protein; MPT, molybdopterin; NR, nitrate reductase; SO, sulfite oxidase; TCEP, Tris(2-carboxyethyl) phosphine hydrochloride; XOR, xanthine oxidoreductase; XO, xanthine oxidase; Mo-enzyme, Moco-containing enzyme.

Research paper thumbnail of Some comparisons between two crystallized anaerobic bacterial rubredoxins from Desulfovibrio gigas and D. vulgaris

Journal of Molecular Biology, 1976

ABSTRACT

Research paper thumbnail of Kinetic studies on the electron transfer between bacterial c-type cytochromes and metal oxides

Journal of Electroanalytical Chemistry, 1998

Cyclic voltammetry was used to investigate the kinetics of the electron transfer between various ... more Cyclic voltammetry was used to investigate the kinetics of the electron transfer between various soluble or solid metal oxides, and polyheme c-type cytochromes from Desulfuromonas acetoxidans and Desulfo6ibrio. The second order rate constant for the catalytic reduction of soluble chromate ions by Desulfuromonas acetoxidans cytochrome c 7 was found to be 6 × 10 5 M − 1 s − 1 . By using the membrane electrode technology, it has been shown that the catalytic process for Cr(VI) reduction is efficient even when the cytochrome is entrapped in the close vicinity of the electrode surface. Moreover, this proceeding allowed the catalytic reduction of solid metal oxides such as manganese(IV), vanadium(V) and iron(III) oxides to be performed. Results suggest that the metal reductase activity of a microorganism is governed by its c-type cytochrome content. Furthermore, only cytochromes with bishistidinyl heme iron coordination act as metal reducers whereas mitochondrial c-type cytochromes do not. This approach opens new pathways for the use of sulfur or sulfate bacteria in the bioremediation of metal contaminated waters and waste streams. Processes involving the use of entrapped enzymes reactors could be developed according to the metal reducing activity of their polyheme c-type cytochromes.

Research paper thumbnail of The amino acid sequence of desulforedoxin, a new type of non heme iron protein from

Biochemical and Biophysical Research Communications, 1979

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

Biochimica et Biophysica Acta (BBA) - Protein Structure and Molecular Enzymology, 1993

Rusticyanin, a copper protein characterized by a high redox potential (+680 mV) and a high stabil... more 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.

Research paper thumbnail of Insight into Molecular Stability and Physiological Properties of the Diheme Cytochrome CYC 41 from the Acidophilic Bacterium Acidithiobacillus ferrooxidans

Biochemistry, 2005

The cyc1 gene encoding the soluble dihemic cytochrome c CYC(41) from Acidithiobacillus ferrooxida... more The cyc1 gene encoding the soluble dihemic cytochrome c CYC(41) from Acidithiobacillus ferrooxidans, an acidophilic organism, has been cloned and expressed in Escherichia coli as the host organism. The cytochrome was successfully produced and folded only in fermentative conditions: this allowed us to determine the molecular basis of the heme insertion at extreme pH. Point mutations at two sequence positions (E121 and Y63) were introduced near the two hemes in order to assign individual redox potentials to the hemes and to identify the interaction sites with the redox partners, rusticyanin and cytochrome oxidase. Characterization of mutants E121A, Y63A, and Y63F CYC(41) with biochemical and biophysical techniques were carried out. Substitution of tyrosine 63 by phenylalanine alters the environment of heme B. This result indicates that heme B has the lower redox potential. Interaction studies with the two physiological partners indicate that CYC(41) functions as an electron wire between RCy and cytochrome oxidase. A specific glutamate residue (E121) located near heme A is directly involved in the interaction with RCy. A docking analysis of CYC(41), RCy, and cytochrome oxidase allowed us to propose a model for the complex in agreement with our experimental data.