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Pseudoazurin (Paz) is the physiological electron donor to copper-containing nitrite reductase (Nir), which catalyzes the reduction of NO2− to NO. The Nir reaction mechanism involves the reduction of the type 1 (T1) copper electron transfer center by the external physiological electron donor, intramolecular electron transfer from the T1 copper center to the T2 copper center, and nitrite reduction at the type 2 (T2) copper catalytic center. We report the cloning, expression, and characterization of Paz from Sinorhizobium meliloti 2011 (SmPaz), the ability of SmPaz to act as an electron donor partner of S. meliloti 2011 Nir (SmNir), and the redox properties of the metal centers involved in the electron transfer chain. Gel filtration chromatography and sodium dodecyl sulfate–polyacrylamide gel electrophoresis together with UV–vis and EPR spectroscopies revealed that as-purified SmPaz is a mononuclear copper-containing protein that has a T1 copper site in a highly distorted tetrahedral geometry. The SmPaz/SmNir interaction investigated electrochemically showed that SmPaz serves as an efficient electron donor to SmNir. The formal reduction potentials of the T1 copper center in SmPaz and the T1 and T2 copper centers in SmNir, evaluated by cyclic voltammetry and by UV-vis- and EPR-mediated potentiometric titrations, are against an efficient Paz T1 center to Nir T1 center to Nir T2 center electron transfer. EPR experiments proved that as a result of the SmPaz/SmNir interaction in the presence of nitrite, the order of the reduction potentials of SmNir reversed, in line with T1 center to T2 center electron transfer being thermodynamically more favorable. Fil: Ferroni, Felix Martín. Universidad Nacional del Litoral. Facultad de Bioquímica y Ciencias Biológicas. Departamento de Física; Argentina. Consejo Nacional de Investigaciones Científicas y Técnicas. Centro Científico Tecnológico Conicet - Santa Fe; Argentina Fil: Marangon, Jacopo. Universidade Nova de Lisboa; Brasil Fil: Neuman, Nicolás Ignacio. Consejo Nacional de Investigaciones Científicas y Técnicas. Centro Científico Tecnológico Conicet - Santa Fe; Argentina. Universidad Nacional del Litoral. Facultad de Bioquímica y Ciencias Biológicas. Departamento de Física; Argentina Fil: Cristaldi, Julio César. Consejo Nacional de Investigaciones Científicas y Técnicas. Centro Científico Tecnológico Conicet - Santa Fe; Argentina. Universidad Nacional del Litoral. Facultad de Bioquímica y Ciencias Biológicas. Departamento de Física; Argentina Fil: Brambilla, Silvina Maricel. Universidad Nacional del Litoral. Facultad de Bioquímica y Ciencias Biológicas. Departamento de Física; Argentina Fil: Guerrero, Sergio Adrian. Consejo Nacional de Investigaciones Científicas y Técnicas. Centro Científico Tecnológico Conicet - Santa Fe. Instituto de Agrobiotecnología del Litoral. Universidad Nacional del Litoral. Instituto de Agrobiotecnología del Litoral; Argentina Fil: Rivas, Maria Gabriela. Consejo Nacional de Investigaciones Científicas y Técnicas. Centro Científico Tecnológico Conicet - Santa Fe; Argentina. Universidade Nova de Lisboa; Brasil. Universidad Nacional del Litoral. Facultad de Bioquímica y Ciencias Biológicas. Departamento de Física; Argentina Fil: Rizzi, Alberto Claudio. Universidad Nacional del Litoral. Facultad de Bioquímica y Ciencias Biológicas. Departamento de Física; Argentina Fil: Brondino, Carlos Dante. Consejo Nacional de Investigaciones Científicas y Técnicas. Centro Científico Tecnológico Conicet - Santa Fe; Argentina. Universidad Nacional del Litoral. Facultad de Bioquímica y Ciencias Biológicas. Departamento de Física; Argentina

Sulfite dehydrogenases are enzymes that catalyze the oxidation of the toxic and mutagenic compound sulfite to sulfate, thereby protecting cells from adverse effects associated with sulfite exposure. While some bacterial sulfite dehydrogenases that have been characterized to date are able to use cytochrome c as an electron acceptor, the majority of these enzymes prefer ferricyanide as an electron acceptor and have therefore been termed ‘atypical’ sulfite dehydrogenases. Identifying the natural electron acceptor of these enzymes, however, is crucial for understanding how the ‘atypical’ sulfite dehydrogenases are integrated into cell metabolism.The SorT sulfite dehydrogenase from Sinorhizobium meliloti is a representative of this enzyme type and we have investigated the interactions of SorT with two small redox proteins, a cytochrome c and a Cu containing pseudoazurin, that are encoded in the same operon and are co-transcribed with the sorT gene. Both potential acceptor proteins have been purified and characterized in terms of their biochemical and electrochemical properties, and interactions and enzymatic studies with both the purified SorT sulfite dehydrogenase and components of the respiratory chain have been carried out.We were able to show for the first time that an ‘atypical’ sulfite dehydrogenase can couple efficiently to a cytochrome c isolated from the same organism despite being unable to efficiently reduce horse heart cytochrome c, however, at present the role of the pseudoazurin in SorT electron transfer is unclear, but it is possible that it acts as an intermediate electron shuttle between. The SorT system appears to couple directly to the respiratory chain, most likely to a cytochrome oxidase.

This work was supported in part by Fundação para a Ciência e a Tecnologia (Lisbon, Portugal) through project grant POCTI/QUI/64638/2006 and individual grant SFRH/BPD/20357/2004 (SN). The authors would like to thank Dr Cecilia Bonifacio for help with crystallization and Dr Jose Trincao for help with data collection. Pseudoazurins are small type 1 copper proteins that are involved in the flow of electrons between various electron donors and acceptors in the bacterial periplasm, mostly under denitrifying conditions. The previously determined structure of Paracoccus pantotrophus pseudoazurin in the oxidized form was improved to a nominal resolution of 1.4 Å, with R and R free values of 0.188 and 0.206, respectively. This high-resolution structure makes it possible to analyze the interactions between the monomers and the solvent structure in detail. Analysis of the high-resolution structure revealed the structural regions that are responsible for monomer-monomer recognition during dimer formation and for protein-protein interaction and that are important for partner recognition. The pseudoazurin structure was compared with other structures of various type 1 copper proteins and these were grouped into families according to similarities in their secondary structure; this may be useful in the annotation of copper proteins in newly sequenced genomes and in the identification of novel copper proteins. publishersversion published

The Cu-containing nitrite reductase from Alcaligenes faecalis S-6 catalyzes the one-electron reduction of nitrite to nitric oxide (NO). Electrons enter the enzyme at the so-called type-1 Cu site and are then transferred internally to the catalytic type-2 Cu site. Protein film voltammetry experiments were carried out to obtain detailed information about the catalytic cycle. The homotrimeric structure of the enzyme is reflected in a distribution of the heterogeneous electron-transfer rates around three main values. Otherwise, the properties and the mode of operation of the enzyme when it is adsorbed as a film on a pyrolytic graphite electrode are essentially unchanged compared to those of the free enzyme in solution. It was established that the reduced type-2 site exists in either an active or an inactive conformation with an interconversion rate of similar to 0.1 s(-1). The random sequential mechanism comprises two routes, one in which the type-2 site is reduced first and subsequently binds nitrite, which is then converted into NO, and another in which the oxidized type-2 site binds nitrite and then accepts an electron to produce NO. At high nitrite concentration, the second route prevails and internal electron transfer is rate-limiting. The midpoint potentials of both sites could be established under catalytic conditions. Binding of nitrite to the type-2 site does not affect the midpoint potential of the type-1 site, thereby excluding cooperativity between the two sites.

The 3D structure of the apo-pseudoazurin (copper free pseudoazurin) from Alcaligenes faecalis strain S-6 is determined and refined at pH 6.7 using X-ray diffraction data to 1.85 A resolution. The final crystallographic R-factor is 0.164. Comparing the structures of apo-pseudoazurin and the native (Cu2+) protein, we observed limited differences ranging between 0.1-0.4 A at the vicinity of the copper site, at the loops connecting the secondary structural elements, at certain beta-strands and at the amino and carboxy termini of the protein.

A periplasmic protein able to transfer electrons from cytoplasmic membrane to the periplasmic nitrite reductase (cytochrome cd1) has been purified from the anoxically grown cytochrome c550 mutant strain Pd2121 and shown to be pseudoazurin by several independent criteria (molecular mass, copper content, visible spectrum, N-terminal amino acid sequence). Under our assay conditions, the half-saturation of electron transport occurred at about 10 microM pseudoazurin; the reaction was retarded by increasing ionic strength.

Kinetic analysis of electron transfer between azurin from Pseudomonas aeruginosa and copper-containing nitrite reductase (NIR) from Alcaligenes faecalis S-6 was carried out to investigate the specificity of electron transfer between copper-containing proteins. Apparent values of kcat and Km of NIR for azurin were 300-fold smaller and 172-fold larger than those for the physiological redox partner, pseudoazurin from A. faecalis S-6, respectively, suggesting that the electron transfer between azurin and NIR was less specific than that between pseudoazurin and NIR. One of the major differences in 3-D structure between these redox proteins, azurin and pseudoazurin, is the absence and presence of lysine residues near their type 1 copper sites, respectively. Three mutated azurins, D11K, P36K, and D11K/P36K, were constructed to evaluate the importance of lysine residues in the interaction with NIR. The redox potentials of D11K, P36K, and D11K/P36K azurins were higher than that of wild-type azurin by 48, 7, and 55 mV, respectively. As suggested by the increase in the redox potential, kinetic analysis of electron transfer revealed reduced ability of electron transfer in the mutated azurins. On the other hand, although each of the single mutations caused modest effects on the decrease in the Km value, the simultaneous mutations of D11K and P36K caused significant decrease in the Km value when compared to that for wild-type azurin. These results suggest that the introduction of two lysine residues into azurin facilitated docking to NIR.

The structures of the reduced (Cu1+) blue-copper protein pseudoazurin from Alcaligenes faecalis strain S-6 are refined at pH 7.8 and 4.4 using X-ray diffraction data to 1.8 A resolution. The final R-factors for the high and low pH structures are 0.178 and 0.177, respectively. Comparing the reduced pseudoazurin at pH 7.8 with the oxidised (Cu2+) molecule, small changes are observed in the vicinity of the copper site and on the protein surface. At pH 4.4 the copper substituent imidazole of His81 rotates away from the metal with a concurrent movement of the latter towards the plane of the remaining three ligands (S gamma-Cys78, N delta 1-His40 and S delta-Met86) thus the geometry of the copper site becomes planar trigonal.