Copper nitrite reductase from Sinorhizobium meliloti 2011: Crystal structure and interaction with the physiological versus a nonmetabolically related cupredoxin-like mediator.

We report the crystal structure of the copper-containing nitrite reductase (NirK) from the Gram-negative bacterium Sinorhizobium meliloti 2011 (Sm), together with complex structural alignment and docking studies with both non-cognate and the physiologically related pseudoazurins, SmPaz1 and SmPaz2, respectively. S. meliloti is a rhizobacterium used for the formulation of Medicago sativa bionoculants, and SmNirK plays a key role in this symbiosis through the denitrification pathway. The structure of SmNirK, solved at a resolution of 2.5 Å, showed a striking resemblance with the overall structure of the well-known Class I NirKs composed of two Greek key ß-barrel domains. The activity of SmNirK is ~12% of the activity reported for classical NirKs, which could be attributed to several factors such as subtle structural differences in the secondary proton channel, solvent accessibility of the substrate channel, and that the denitrifying activity has to be finely regulated within the endosymbiont. In vitro kinetics performed in homogenous and heterogeneous media showed that both SmPaz1 and SmPaz2, which are coded in different regions of the genome, donate electrons to SmNirK with similar performance. Even though the energetics of the interprotein electron transfer (ET) process is not favorable with either electron donors, adduct formation mediated by conserved residues allows minimizing the distance between the copper centers involved in the interprotein ET process.

The Zero-Order Loop in Apoazurin Modulates Folding Mechanism In Silico.

Pseudomonas aeruginosa apoazurin (apo, without the copper cofactor) has a single disulfide bond between residues 3 and 26 and unfolds in a two-state reaction in vitro. The disulfide bond covalently connects the N-termini of ß-strands 1 and 3; thereby, it creates a zero-order loop or a "cinch" that restricts conformational space. Covalent loops and threaded topologies are emerging as important structural elements in folded proteins and may be important for function. In order to understand the role of a zero-order loop in the folding process of a protein, here we used coarse-grained molecular dynamics (CGMD) simulations in silico to compare two variants of apoazurin: one named "loop" which contained the disulfide, and another named "open" in which the disulfide bond between residues 3 and 26 was removed. CGMD simulations were performed to probe the stability and unfolding pathway of the two apoazurin variants at different urea concentrations and temperatures. Our results show that the covalent loop plays a prominent role in the unfolding mechanism of apoazurin; its removal alters both the folding-transition state and the unfolded-state ensemble of conformations. We propose that modulation of azurin's folding landscape by the disulfide bridge may be related to both copper capturing and redox sensing.

Pseudoazurin from Sinorhizobium meliloti as an electron donor to copper-containing nitrite reductase: influence of the redox partner on the reduction potentials of the enzyme copper centers.

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.

Inferring a weighted elastic network from partial unfolding with coarse-grained simulations.

A number of studies have demonstrated that simple elastic network models can reproduce experimental B-factors, providing insights into the structure-function properties of proteins. Here, we report a study on how to improve an elastic network model and explore its performance by predicting the experimental B-factors. Elastic network models are built on the experimental Cα coordinates, and they only take the pairs of Cα atoms within a given cutoff distance rc into account. These models describe the interactions by elastic springs with the same force constant. We have developed a method based on numerical simulations with a simple coarse-grained force field, to attribute weights to these spring constants. This method considers the time that two Cα atoms remain connected in the network during partial unfolding, establishing a means of measuring the strength of each link. We examined two different coarse-grained force fields and explored the computation of these weights by unfolding the native structures.

Flexibility of the metal-binding region in apo-cupredoxins.

Protein-mediated electron transfer is an essential event in many biochemical processes. Efficient electron transfer requires the reorganization energy of the redox event to be minimized, which is ensured by the presence of rigid donor and acceptor sites. Electron transfer copper sites are present in the ubiquitous cupredoxin fold, able to bind one or two copper ions. The low reorganization energy in these metal centers has been accounted for by assuming that the protein scaffold creates an entatic/rack-induced state, which gives rise to a rigid environment by means of a preformed metal chelating site. However, this notion is incompatible with the need for an exposed metal-binding site and protein-protein interactions enabling metallochaperone-mediated assembly of the copper site. Here we report an NMR study that reveals a high degree of structural heterogeneity in the metal-binding region of the nonmetallated Cu(A)-binding cupredoxin domain, arising from microsecond to second dynamics that are quenched upon metal binding. We also report similar dynamic features in apo-azurin, a paradigmatic blue copper protein, suggesting a general behavior. These findings reveal that the entatic/rack-induced state, governing the features of the metal center in the copper-loaded protein, does not require a preformed metal-binding site. Instead, metal binding is a major contributor to the rigidity of electron transfer copper centers. These results reconcile the seemingly contradictory requirements of a rigid, occluded center for electron transfer, and an accessible, dynamic site required for in vivo copper uptake.

The rus operon genes are differentially regulated when Acidithiobacillus ferrooxidans LR is kept in contact with metal sulfides.

Acidithiobacillus ferrooxidans is a gram-negative bacterium that obtains energy from the oxidation of ferrous iron or reduced sulfur compounds. In this bacterium, the proteins encoded by the rus operon are involved in electron transfer from Fe(II) to O(2), and the first two proteins in this pathway also participate in the electron transfer pathway from Fe(II) to NAD(P). In this work we analyzed the expression, by real-time PCR, of the eight genes from the rus operon when A. ferrooxidans LR was grown in the presence of iron (control) and then kept in contact with chalcopyrite (CuFeS(2)) and covellite (CuS). A small decrease in rus operon gene expression was observed in the presence of chalcopyrite, while in the presence of covellite the expression of these genes showed a remarkable decrease. These results can be explained by the absence of ferrous iron in covellite. To explain the expression difference observed between the gene cyc1 and the gene rus, we investigated the information content presented at the Translation Initiation Site (TIS) of both genes. cyc1 showed a highly information content (8.4 bits) that can maximize translation, and rus showed a less favorable context (5.5 bits). Our hypothesis is that the energetic metabolism in A. ferrooxidans may be controlled at the transcriptional and posttranscriptional level by different mechanisms.

Regulation of the expression of the Acidithiobacillus ferrooxidans rus operon encoding two cytochromes c, a cytochrome oxidase and rusticyanin.

The regulation of the expression of the rus operon, proposed to encode an electron transfer chain from the outer to the inner membrane in the obligate acidophilic chemolithoautroph Acidithiobacillus ferrooxidans, has been studied at the RNA and protein levels. As observed by Northern hybridization, real-time PCR and reverse transcription analyses, this operon was more highly expressed in ferrous iron- than in sulfur-grown cells. Furthermore, it was shown by immunodetection that components of this respiratory chain are synthesized in ferrous iron- rather than in sulfur-growth conditions. Nonetheless, weak transcription and translation products of the rus operon were detected in sulfur-grown cells at the early exponential phase. The results strongly support the notion that rus-operon expression is induced by ferrous iron, in agreement with the involvement of the rus-operon-encoded products in the oxidation of ferrous iron, and that ferrous iron is used in preference to sulfur.

Metal-ligand interplay in blue copper proteins studied by 1H NMR spectroscopy: Cu(II)-pseudoazurin and Cu(II)-rusticyanin.

The blue copper proteins (BCPs), pseudoazurin from Achromobacter cycloclastes and rusticyanin from Thiobacillus ferrooxidans, have been investigated by (1)H NMR at a magnetic field of 18.8 T. Hyperfine shifts of the protons belonging to the coordinated ligands have been identified by exchange spectroscopy, including the indirect detection for those resonances that cannot be directly observed (the beta-CH(2) of the Cys ligand, and the NH amide hydrogen bonded to the S(gamma)(Cys) atom). These data reveal that the Cu(II)-Cys interaction in pseudoazurin and rusticyanin is weakened compared to that in classic blue sites (plastocyanin and azurin). This weakening is not induced by a stronger interaction with the axial ligand, as found in stellacyanin, but might be determined by the protein folding around the metal site. The average chemical shift of the beta-CH(2) Cys ligand in all BCPs can be correlated to geometric factors of the metal site (the Cu-S(gamma)(Cys) distance and the angle between the CuN(His)N(His) plane and the Cu-S(gamma)(Cys) vector). It is concluded that the degree of tetragonal distortion is not necessarily related to the strength of the Cu(II)-S(gamma)(Cys) bond. The copper-His interaction is similar in all BCPs, even for the solvent-exposed His ligand. It is proposed that the copper xy magnetic axes in blue sites are determined by subtle geometrical differences, particularly the orientation of the His ligands. Finally, the observed chemical shifts for beta-CH(2) Cys and Ser NH protons in rusticyanin suggest that a less negative charge at the sulfur atom could contribute to the high redox potential (680 mV) of this protein.

Alkaline transition of Rhus vernicifera stellacyanin, an unusual blue copper protein.

Stellacyanin from Rhus vernificera is a blue copper protein in which the metal is coordinated to a Cys, two His, and a Gln residue. It displays a low redox potential, a fast electron exchange rate, and a reversible alkaline transition. We have studied this transition in Cu(II)- and Co(II)-stellacyanin by means of electronic and NMR spectroscopy. The data indicate that a conformational rearrangement of the metal site occurs at high pH. A drastic alteration in the Gln coordination mode, as initially proposed, is discarded. These results show that the metal site in stellacyanin is more flexible than the sites of other blue copper proteins. The present study demonstrates that the paramagnetic shifts of the bound Cys in the Co(II) derivative are sensitive indicators of the electron delocalization and conformational changes experienced by this residue.

Histochemical staining of bone aluminum: comparison of aluminon and acid solochrome azurine and their correlation with bone aluminum content

O aluminio (Al) pode ser um dos fatores patogenicos envolvidos na doença ossea de pacientes uremicos. Os corantes Aluminon (Acido Tricarboxilico aurico) e Acido Solocromo Azurina, tem sido utilizados para detectar depositos de aluminio no tecido osseo. Utilizamos um modelo experimental de intoxicaçäo aluminica em ratos normais (N) e uremicos (U) e comparamos a sensibilidade dos dois corantes na detecçäo do aluminio. Os grupos receberam injeçöes intraperitoniais de Cloreto de Aluminio (AlCl3), ate uma dose cumulativa de 5 mg (NAL5; UAL5) e 30 mg (NAL30; UAL30). Os grupos controles receberam injeçöes intraperitoniais de agua destilada...

Structural fluctuations in proteins revealed by time-resolved fluorescence spectroscopy.

Time-resolved fluorescence spectroscopy permits the direct assessment of proteins motions in the picosecond-nanosecond time-scaled, i.e., in a time-window compatible with observation of relevant motions of the protein matrix. The intrinsic fluorescence emission from tryptophan and tyrosine residues provides a convenient tool to follow these dynamic events in proteins. In the present investigation, the use of time-resolved fluorescence spectroscopy to monitor protein dynamics is illustrated by a study of the effects of temperature and calcium binding on the internal dynamics of the calcium-binding protein, parvalbumin, and by an investigation of the effects of hydration on the measurements of both fluorescence intensity and anisotropy decays provided complementary information regarding the flexibility of aromatic side chains in the proteins investigated, which could be correlated with environmental effects on protein structure.