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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 β‐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. PDB Code(s): 7P2F; [ABSTRACT FROM AUTHOR]

Agrobacterium tumefaciens S33 can grow with nicotine as the sole source of carbon, nitrogen, and energy via a novel hybrid of the pyridine pathway and the pyrrolidine pathway. Characterization of the enzymes involved in the hybrid pathway is important for understanding its biochemical mechanism. Here, we report that the molybdenum-containing nicotine dehydrogenase (NdhAB), which catalyzes the initial step of nicotine degradation, is located in the periplasm of strain S33, while the 6-hydroxynicotine oxidase and 6-hydroxypseudooxynicoine oxidase are in the cytoplasm. This is consistent with the fact that NdhA has a Tat signal peptide. Interestingly, an open reading frame (ORF) adjacent to the ndhAB gene was verified to encode a copper-containing electron carrier, pseudoazurin (Paz), which has a signal peptide typical of bacterial Paz proteins. Both were transported into the periplasm after being produced in the cytoplasm. We purified NdhAB from the periplasmic fraction of strain S33 and found that with Paz as the physiological electron acceptor, NdhAB catalyzed the hydroxylation of nicotine at a specific rate of 110.52 ± 8.09 ±μmol ∙ min-1 ∙ mg of protein-1, where the oxygen atom in the hydroxyl group of the product 6-hydroxynicotine was derived from H2O. The apparent Km values for nicotine and Paz were 1.64 ± 0.07 ±μM and 3.61 ± 0.23 ±M, respectively. NAD(P)+, O2, and ferredoxin could not serve as electron acceptors. Disruption of the paz gene disabled the strain for nicotine degradation, indicating that Paz is required for nicotine catabolism in the strain. These findings help our understanding of electron transfer during nicotine degradation in bacteria. IMPORTANCE Nicotine is a toxic and addictive N-heterocyclic aromatic alkaloid produced in tobacco. Its catabolism in organisms and degradation in tobacco wastes have become major concerns for human health and the environment. Bacteria usually decompose nicotine using the classical strategy of hydroxylating the pyridine ring with the help of activated oxygen by nicotine dehydrogenase, which binds one molybdopterin, two [2Fe2S] clusters, and usually one flavin adenine dinucleotide (FAD) as well. However, the physiological electron acceptor for the reaction is still unknown. In this study, we found that the two-component nicotine dehydrogenase from Agrobacterium tumefaciens S33, naturally lacking an FAD-binding domain, is located in the periplasmic space and uses a copper-containing electron carrier, pseudoazurin, as its physiological electron acceptor. We report here the role of pseudoazurin in a reaction catalyzed by a molybdopterin-containing hydroxylase occurring in the periplasmic space. These results provide new biochemical knowledge on microbial degradation of N-heterocyclic aromatic compounds. [ABSTRACT FROM AUTHOR]

Pseudoazurin from the denitrifying bacterium Alcaligenes faecalis ( AfPAz) is a blue copper protein and functions as an electron donor to copper-containing nitrite reductase (CuNIR). Conventionally, AfPAz has been crystallized using highly concentrated ammonium sulfate as a precipitant. Here, a needle-like crystal of AfPAz grown in a solution containing a macromolecular precipitant, polyethylene glycol 8000 (PEG 8000), is reported. The crystal belonged to space group P61, with unit-cell parameters a = b = 68.7, c = 94.2 Å. The structure has been determined and refined at 2.6 Å resolution. The asymmetric unit contained two AfPAz molecules contacting each other on negatively charged surfaces. The molecular packing of the crystal showed a right-handed double-helical arrangement of AfPAz molecules and hence of blue copper sites. This structure provides insight into the excluded-volume effect of PEG and the manner of assembly of AfPAz. [ABSTRACT FROM AUTHOR]

The copper(II) centre of the blue copper protein pseudoazurin from Alcaligenes faecalis has been substituted by zinc(II) via denaturing the protein, chelation and removal of copper and refolding the apoprotein, followed by the addition of an aqueous solution of ZnCl2. Vapour-diffusion experiments produced colourless hexagonal crystals (space group P65), which when cryocooled had unit-cell parameters a = b = 49.01, c = 98.08 Å. Diffraction data collected at 100 K using a copper sealed tube were phased by the weak anomalous signal of five S atoms and one Zn atom. The structure was fitted manually and refined to 1.6 Å resolution. The zinc-substituted protein exhibits similar overall geometry to the native structure with copper. Zn2+ binds more strongly to its four ligand atoms (His40 Nδ1, Cys78 Sγ, His81 Nδ1 and Met86 Sδ) and retains the tetrahedral arrangement, although the structure is less distorted than the native copper protein. [ABSTRACT FROM AUTHOR]

Pseudoazurin (Paz) is the physiological electron donor to copper-containing nitrite reductase (Nir), which catalyzes the reduction of NO 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. [ABSTRACT FROM AUTHOR]

Abstract: We report on a high-frequency electron-paramagnetic-resonance study of the type 1 copper site of pseudoazurin. The spectra fully resolve the contribution of a nearly axial spectrum besides the rhombic spectrum, which unequivocally proves the existence of two conformations of the copper site. Pseudoazurins have been considered from Achromobacter cycloclastes including eight mutants and from Alcaligenes faecalis. The two conformations are virtually the same for all pseudoazurins, but the rhombic/axial population varies largely, between 91/9 and 33/67. These observations are discussed in relation to optical absorption spectra and X-ray diffraction structures. A similar observation for fern plastocyanin from Dryopteris crassirhizoma suggests that dual conformations of type 1 copper sites are more common. [Copyright &y& Elsevier]

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.

The use of paramagnetic NMR data for the refinement of structures of proteins and protein complexes is widespread. However, the power of paramagnetism for protein assignment has not yet been fully exploited. PARAssign is software that uses pseudocontact shift data derived from several paramagnetic centers attached to the protein to obtain amide and methyl assignments. The ability of PARAssign to perform assignment when the positions of the paramagnetic centers are known and unknown is demonstrated. PARAssign has been tested using synthetic data for methyl assignment of a 47 kDa protein, and using both synthetic and experimental data for amide assignment of a 14 kDa protein. The complex fitting space involved in such an assignment procedure necessitates that good starting conditions are found, both regarding placement and strength of paramagnetic centers. These starting conditions are obtained through automated tensor placement and user-defined tensor parameters. The results presented herein demonstrate that PARAssign is able to successfully perform resonance assignment in large systems with a high degree of reliability. This software provides a method for obtaining the assignments of large systems, which may previously have been unassignable, by using 2D NMR spectral data and a known protein structure. [ABSTRACT FROM AUTHOR]

Abstract: The X-ray crystal structure of oxidised pseudoazurin from the denitrifying plant symbiotic bacterium Sinorhizobium meliloti (SmPAz2) has been solved to a resolution of 2.0Å. The pseudoazurin from Sinorhizobium sp. is unusual as it forms an operon with a sulfite dehydrogenase enzyme, rather than a Cu nitrite reductase. Examination of the structure reveals that the geometric parameters of the Type I Cu site in SmPAz2 correlate with observed features in the electronic spectrum of the protein. Comparison of the structure of SmPAz2 with those of pseudoazurins from five other bacterial species shows that the surface of SmPAz2 bears a conserved hydrophobic patch encircled by positively-charged residues, which may serve as a recognition site for its redox partners. [Copyright &y& Elsevier]

Abstract: The short Cu2+–S(Met) bond in pseudoazurin (PAz) results in the presence of two relatively intense Sp(π) and Sp(σ) charge transfer (CT) transitions. This has enabled resonance Raman (rR) data to be obtained for each excited state. The rR data show very different intensity distribution patterns for the vibrations in the 300–500cm−1 region. Time-dependent density functional theory (TDDFT) calculations have been used to determine that the change in intensity distribution between the Sp(π) and Sp(σ) excited states reflects the differential enhancement of S(Cys) backbone modes with Cu–S(Cys)–Cβ out-of-plane (oop) and in-plane (ip) bend character in their respective potential energy distributions (PEDs). The rR excited state distortions have been related to ground state reorganization energies (λs) and predict that, in addition to M–L stretches, the Cu–S(Cys)–Cβ oop bend needs to be considered. DFT calculations predict a large distortion in the Cu–S(Cys)–Cβ oop bending coordinate upon reduction of a blue copper (BC) site; however, this distortion is not present in the X-ray crystal structures of reduced BC sites. The lack of Cu–S(Cys)–Cβ oop distortion upon reduction corresponds to a previously unconsidered constraint on the thiolate ligand orientation in the reduced state of BC proteins and can be considered as a contribution to the entatic/rack nature of BC sites. [Copyright &y& Elsevier]

Abstract: Pseudoazurin (PAz), a well-characterized blue copper electron-transfer protein, is shown herein to be capable of mediating electron transfer to the nitrous oxide reductase (N2OR) from Achromobacter cycloclastes (Ac). Spectroscopic measurements demonstrate that reduced PAz is efficiently re-oxidized by a catalytic amount of N2OR in the presence of N2O. Fits of the kinetics resulted in K M (N2O) and k cat values of 19.1±3.8μM and 89.3±4.2s−1 respectively. The K M (PAz) was 28.8±6.6μM. The electrochemistry of Ac pseudoazurin (AcPAz) in the presence of Ac nitrous oxide reductase (AcN2OR) and N2O displayed an enhanced cathodic sigmoidal current–potential curve, in excellent agreement with the re-oxidation of reduced AcPAz during the catalytic reduction of N2O by AcN2OR. Modeling the structure of the AcPAz–AcN2OR electron transfer complex indicates that AcPAz binds near CuA in AcN2OR, with parameters consistent with the formation of a transient, weakly-bound complex. Multiple, potentially efficient electron-transfer pathways between the blue-copper center in AcPAz and CuA were also identified. Collectively, the data establish that PAz is capable of donating electrons to N2OR in N2O reduction and is a strong candidate for the physiological electron donor to N2OR in Ac. [Copyright &y& Elsevier]

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 Rfree 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. [ABSTRACT FROM AUTHOR]

A method for the electrical contacting of redox enzymes that obtain oxidizing or reducing equivalents from small electron-transfer proteins is demonstrated. The electrochemical contacting of redox enzymes through their immobilization onto electrode supports offers great potential for technological applications and for fundamental studies, but finding appropriate methods to immobilize the enzymes in an orientation allowing rapid electron transfer with the electrode has proven difficult. The copper enzyme nitrite reductase (NiR) and its natural electron-exchange partner pseudoazurin (Paz) are conjugated to a specific DNA tag and immobilized to a gold electrode into a stoichiometrically defined assembly. The DNA tethered to the electrode surface acts as flexible place-holder for the protein components, allowing both proteins to move within the construct. It is shown that Paz efficiently shuttles electrons between the electrode and the NiR enzyme, allowing the electrochemically driven NiR catalysis to be monitored. The activity of the NiR enzyme remains unperturbed by the immobilization. The rate-limiting step of the system is tentatively ascribed to the dissociation of the Paz/NiR complex. The electrochemical response of the system reports not only on the NiR catalysis and on interfacial electron transfer but also on the interaction between NiR and Paz. [ABSTRACT FROM AUTHOR]

Abstract: We have used low-temperature (77K) resonance Raman (RR) spectroscopy as a probe of the electronic and molecular structure to investigate weak π–π interactions between the metal ion-coordinated His imidazoles and aromatic side chains in the second coordination sphere of blue copper proteins. For this purpose, the RR spectra of Met16 mutants of Achromobacter cycloclastes pseudoazurin (AcPAz) with aromatic (Met16Tyr, Met16Trp, and Met16Phe) and aliphatic (Met16Ala, Met16Val, Met16Leu, and Met16Ile) amino acid side chains have been obtained and analyzed over the 100–500cm−1 spectral region. Subtle strengthening of the Cu(II)–S(Cys) interaction on replacing Met16 with Tyr, Trp, and Phe is indicated by the upshifted (0.3–0.8cm−1) RR bands involving ν(Cu–S)Cys stretching modes. In contrast, the RR spectra of Met16 mutants with aliphatic amino acids revealed larger (0.2–1.8cm−1) shifts of the ν(Cu–S)Cys stretching modes to a lower frequency region, which indicate a weakening of the Cu(II)–S(Cys) bond. Comparisons of the predominantly ν(Cu–S)Cys stretching RR peaks of the Met16X=Tyr, Trp, and Phe variants, with the molar absorptivity ratio ε 1/ε 2 of σ(∼455nm)/π(∼595nm) (Cys)S→Cu(II) charge-transfer bands in the optical spectrum and the axial/rhombic EPR signals, revealed a slightly more trigonal disposition of ligands about the copper(II) ion. In contrast, the RR spectra of Met16Z=Ala, Val, Leu, and Ile variants with aliphatic amino acid side chains show a more tetrahedral perturbation of the copper active site, as judged by the lower frequencies of the ν(Cu–S)Cys stretching modes, much larger values of the ε 1/ε 2 ratio, and the increased rhombicity of the EPR spectra. [Copyright &y& Elsevier]

Abstract: The blue or Type 1 (T1) copper site of Paracoccus pantotrophus pseudoazurin exhibits significant absorption intensity in both the 450 and 600nm regions. These are σ and π SCys to Cu2+ charge transfer (CT) transitions. The temperature dependent absorption, EPR, and resonance Raman (rR) vibrations enhanced by these bands indicate that a single species is present at all temperatures. This contrasts the temperature dependent behavior of the T1 center in nitrite reductase [S. Ghosh, X. Xie, A. Dey, Y. Sun, C. Scholes, E. Solomon, Proc. Natl. Acad. Sci. 106 (2009) 4969–4974] which has a thioether ligand that is unconstrained by the protein. The lack of temperature dependence in the T1 site in pseudoazurin indicates the presence of a protein constraint similar to the blue Cu site in plastocyanin where the thioether ligand is constrained at 2.8 Å. However, plastocyanin exhibits only π CT. This spectral difference between pseudoazurin and plastocyanin reflects a coupled distortion of the site where the axial thioether in pseudoazurin is also constrained, but at a shorter Cu–SMet bond length. This leads to an increase in the Cu2+–SCys bond length, and the site undergoes a partial tetragonal distortion in pseudoazurin. Thus, its ground state wavefunction has both σ and π character in the Cu2+–SCys bond. [Copyright &y& Elsevier]

The complex between Cu-containing nitrite reductase (HdNIR) and its electron-donor protein pseudoazurin (HdPAz) from Hyphomicrobium denitrificans has been crystallized. The crystals were obtained from a mixture of the two proteins using the hanging-drop vapour-diffusion method in the presence of polyethylene glycol (PEG) and 2-methyl-2,4-pentanediol (MPD) as precipitants. SDS-PAGE analysis demonstrated that the crystals contained both proteins. The X-ray diffraction experiment was carried out at SPring-8 and diffraction data were collected to 3.3 Å resolution. The crystals were tetragonal (space group P41212), with unit-cell parameters a = b = 130.39, c = 505.55 Å. Preliminary analysis indicated that there was one HdNIR and at least two HdPAz molecules in the asymmetric unit of the crystal. [ABSTRACT FROM AUTHOR]

Cytochrome cd1 is a respiratory nitrite reductase found in the periplasm of denitrifying bacteria. When fully reduced Paracoccus pantotrophus cytochrome cd1 is mixed with nitrite in a stopped-flow apparatus in the absence of excess reductant, a kinetically stable complex of enzyme and product forms, assigned as a mixture of cFe(II) d1Fe(II)-NO+ and cFe(III) d1Fe(II)-NO (cd1-X). However, in order for the enzyme to achieve steady-state turnover, product (NO) release must occur. In this work, we have investigated the effect of a physiological electron donor to cytochrome cd1, the copper protein pseudoazurin, on the mechanism of nitrite reduction by the enzyme. Our data clearly show that initially oxidized pseudoazurin causes rapid further turnover by the enzyme to give a final product that we assign as all-ferric cytochrome cd1 with nitrite bound to the d1 heme (i.e. from which NO had dissociated). Pseudoazurin catalyzed this effect even when present at only one-tenth the stoichiometry of cytochrome cd1. In contrast, redox-inert zinc pseudoazurin did not affect cd1-X, indicating a crucial role for electron movement between monomers or individual enzyme dimers rather than simply a protein-protein interaction. Furthermore, formation of cd1-X was, remarkably, accelerated by the presence of pseudoazurin, such that it occurred at a rate consistent with cd1-X being an intermediate in the catalytic cycle. It is clear that cytochrome cd1 functions significantly differently in the presence of its two substrates, nitrite and electron donor protein, than in the presence of nitrite alone. [ABSTRACT FROM AUTHOR]

Abstract: Several blue copper proteins are known to change the active site structure at alkaline pH (alkaline transition). Spectroscopic studies of Met16Phe, Met16Tyr, Met16Trp, and Met16Val pseudoazurin variants were performed to investigate the second sphere role through alkaline transition. The visible electronic absorption and resonance Raman spectra of Met16Phe, Met16Tyr, and Met16Trp variants showed the increasing of axial component at pH ∼11 like wild-type PAz. The visible electronic absorption and far-UV CD spectra of Met16Val demonstrated that the destabilization of the protein structure was triggered at pH>11. Resonance Raman (RR) spectra of PAz showed that the intensity-weighted averaged Cu–S(Cys) stretching frequency was shifted to higher frequency region at pH ∼11. The higher frequency shift of Cu–S(Cys) bond is implied the stronger Cu–S(Cys) bond at alkaline transition pH ∼11. The visible electronic absorption and far-UV CD spectra of Met16X PAz revealed that the Met16Val variant is denatured at pH>11, but Met16Phe, Met16Tyr, and Met16Trp mutant proteins are not denatured even at pH>11. These observations suggest that Met16 is important to maintain the protein structure through the possible weak interaction between methionine –SCH3 part and coordinated histidine imidazole moiety. The introduction of π–π interaction in the second coordination sphere may be contributed to the enhancement of protein structure stability. [Copyright &y& Elsevier]

Abstract: Copper-containing nitrite reductase is able to catalyze the reduction of nitrite with a turnover rate of several hundreds per second. Electrons for the reaction are donated by the electron transfer protein pseudoazurin. The process of protein complex formation, electron transfer and dissociation must occur on the millisecond timescale to enable the fast turnover of the enzyme. The structure of this transient protein complex has been studied using paramagnetic NMR spectroscopy. Gadolinium complexes were attached specifically through two engineered Cys residues on three sites on the surface of nitrite reductase, causing strong distance-dependent relaxation effects on the residues of pseudoazurin. Docking of the two proteins based on these NMR-derived distance restraints and the chemical shift perturbation data shows convergence to a cluster of structures with an average root-mean-square deviation of 1.5 Å. The binding interface consists of polar and non-polar residues surrounded by charges. The interprotein distance between the two type-1 copper sites is 15.5(±0.5) Å, enabling fast interprotein electron transfer. The NMR-based lower limit estimate of 600 s−1 for the dissociation rate constant and the fast electron transfer are consistent with the transient nature of the complex. [Copyright &y& Elsevier]

This work reports the direct electrochemistry of Paracoccus pantotrophus pseudoazurin and the mediated catalysis of cytochrome c peroxidase from the same organism. The voltammetric behaviour was examined at a gold membrane electrode, and the studies were performed in the presence of calcium to enable the peroxidase activation. A formal reduction potential, E 0′, of 230 ± 5 mV was determined for pseudoazurin at pH 7.0. Its voltammetric signal presented a pH dependence, defined by p K values of 6.5 and 10.5 in the oxidised state and 7.2 in the reduced state, and was constant up to 1 M NaCl. This small copper protein was shown to be competent as an electron donor to cytochrome c peroxidase and the kinetics of intermolecular electron transfer was analysed. A second-order rate constant of 1.4 ± 0.2 × 105 M−1 s−1 was determined at 0 M NaCl. This parameter has a maximum at 0.3 M NaCl and is pH-independent between pH 5 and 9. [ABSTRACT FROM AUTHOR]

Noncovalent weak interactions play important roles in biological systems. In particular, such interactions in the second coordination shell of metal ions in proteins may modulate the structure and reactivity of the metal ion site in functionally significant ways. Recently, π– π interactions between metal ion coordinated histidine imidazoles and aromatic amino acids have been recognized as potentially important contributors to the properties of metal ion sites. In this paper we demonstrate that in pseudoazurin (a blue copper protein) the π– π interaction between a coordinated histidine imidazole ring and the side chains of aromatic amino acids in the second coordination sphere, significantly influences the properties of the blue copper site. Electronic absorption and electron paramagnetic resonance spectra indicate that the blue copper electronic structure is perturbed, as is the redox potential, by the introduction of a second coordination shell π– π interaction. We suggest that the π– π interaction with the metal ion coordinated histidine imidazole ring modulates the electron delocalization in the active site, and that such interactions may be functionally important in refining the reactivity of blue copper sites. [ABSTRACT FROM AUTHOR]

Bacterial copper-containing nitrite reductase catalyzes the reduction of nitrite to nitric oxide as part of the denitrification process. Pseudoazurin interacts with nitrite reductase in a transient fashion to supply the necessary electrons. The redox-state dependence of complex formation between pseudoazurin and nitrite reductase was studied by nuclear magnetic resonance spectroscopy and isothermal titration calorimetry. Binding of pseudoazurin in the reduced state is characterized by the presence of two binding modes, a slow and a fast exchange mode, with a Kdapp of 100 µM. In the oxidized state of pseudoazurin, binding occurs in a single fast exchange mode with a similar affinity. Metal-substituted proteins have been used to show that the mode of binding of pseudoazurin is independent of the metal charge of nitrite reductase. Contrary to what was found for other cupredoxins, protonation of the exposed His ligand to the copper of pseudoazurin, His81, does not appear to be involved directly in the dual binding mode of the reduced form. A model assuming the presence of a minor form of pseudoazurin is proposed to explain the behavior of the complex in the reduced state. [ABSTRACT FROM AUTHOR]

An extended kinetic model is presented for the process catalysed by two enzymes mutually connected by the cycling of two reversibly interconvertible chemically relative species. Expressions are derived for the steady-state velocity, limiting velocity (V) and the half-saturation concentration of the cycling substrate (A0.5). It is shown that the velocity depends on the total concentration of cycling substrate hyperbolically if both enzymes have equal activities. Based on these theoretical considerations, an experimental comparison was made between pseudoazurin and cytochrome c550 as physiological electron transfer mediators for nitrite reduction in an in vitro reconstituted part of the respiratory chain of Paracoccus denitrificans. Pseudoazurin exhibited 1.7-fold higher V and 14-fold higher A0.5 than cytochrome c550 under the experimental conditions used (20 mM Tris chloride, pH 7.3, 30 °C). [Copyright &y& Elsevier]

Pseudoazurin is an electron transfer copper protein, a member of the cupredoxin family. The protein is frequently found in denitrifying bacteria, where it is the electron donor of nitrite reductase. The copper at the active site is coordinated to His40, Cys78, His81 and Met86 in a distorted tetragonal geometry. We have recorded and assigned the 1H NMR spectra of Co(II)-substituted pseudoazurin from Achromobacter cycloclastes. The 1H NMR spectrum of Co(II)-pseudoazurin closely resembles that of Co(II)-rusticyanin, reflecting an altered conformation for the Met-Co(II)-Cys moiety in both proteins, compared to Co(II)-azurin, amicyanin and stellacyanin. The electron spin density onto the Sγ(Cys) is larger in Co(II)-pseudoazurin compared to Co(II)-rusticyanin. Instead, the Co(II)-Met interaction is similar in both derivatives. Hence, the different metal-ligand interactions might be independently modulated by the protein structure. The present work also shows that the electron spin density onto the Co(II)-Scys bond is sensibly smaller than the Cu(II)-Scys. Notwithstanding, NMR data on Co(II)-substituted blue copper proteins can be safely extrapolated to native Cu(II) proteins. [ABSTRACT FROM AUTHOR]

UV resonance Raman (UVRR) and 1H NMR spectra are measured for native Cu(I)– and Cu(II)–pseudoazurin, its apo-protein, and a few metal-substituted derivatives. The pH titration experiments of 1H NMR enabled us to determine the pKa* values of three His residues (His6, His40, and His81). The UVRR band characteristic of a metal coordinated histidyl imidazole was observed at 1385 cm−1 for Cu(II)–pseudoazurin in D2O but not for Cu(I)–pseudoazurin. This frequency is consistent with the Nδ coordination of His. For the Cu(I)–pseudoazurin a characteristic band of histidyl imidazolium was detected at 1408 cm−1 at acidic pH. This is assigned to protonated His81, which is deligated from Cu(I) at low pH values. The imidazolium Raman band at 1408 cm−1 was also detected in the UVRR spectrum of apo-pseudoazurin at pH* 3.9, and the acidification was accompanied by a significant change in the X-Pro bands at 1467 cm−1. Pseudoazurin substituted with Zn2+ and Cd2+ gave the characteristic Raman bands of the metal coordinated imidazole at 1388 and 1384 cm−1, respectively, at neutral pH, but its intensity diminished upon lowering the pH, and instead the imidazolium band at 1408 cm−1 grew. The X-Pro bands of the pseudoazurins substituted with Zn2+ and Cd2+ exhibited the pH dependence very similar to that observed for apo-pseudoazurin. These findings indicate that the Zn2+ and Cd2+ ions are released from the active site at acidic pH and it is accompanied by a change in hydrogen bonding state of Pro80. This behavior is clearly absent in both the Cu(I) and Cu(II) proteins, meaning that pseudoazurin discriminates between copper and the other metal ions. [Copyright &y& Elsevier]

We have applied scanning tunnelling microscopy (STM) to the study of two proteins: pseudoazurin and apo-pseudoazurin. Both proteins adsorbed onto a Au (1 1 1) surface are visible to STM individually, forming into layers and multilayers, with currents from about 55 to 600 pA. The images reproduce well the expected dimensions laterally but not in the z direction. The apparent height of the proteins varies with the voltage polarity, being higher at negative sample voltages. The bias also affects their shape. Negative sample voltages of more than 1.5 V orient the proteins present on a gold terrace in parallel rows. The layer of water adsorbed on surfaces in ambient conditions can be related to our results to explain the reduced z dimensions, the asymmetry with the voltage polarity and the alignment of proteins at voltages more negative than −1.5 V. [Copyright &y& Elsevier]

The folding of apo-pseudoazurin, a 123-residue, predominantly β-sheet protein with a complex Greek key topology, has been investigated using several biophysical techniques. Kinetic analysis of refolding using far- and near-ultraviolet circular dichroism (UV CD) shows that the protein folds slowly to the native state with rate constants of 0.04 and 0.03 min−1, respectively, at pH 7.0 and at 15°C. This process has an activation enthalpy of ∼90 kJ/mole and is catalyzed by cyclophilin A, indicating that folding is limited by trans- cis proline isomerization, presumably around the Xaa-Pro 20 bond that is in the cis isomer in the native state. Before proline isomerization, an intermediate accumulates during folding. This species has a substantial signal in the far-UV CD, a nonnative signal in the near-UV CD, exposed hydrophobic surfaces (judged by 1-anilino naphthalenesulphonate binding), a noncooperative denaturation transition, and a dynamic structure (revealed by line broadening on the nuclear magnetic resonance time scale). We compare the properties of this intermediate with partially folded states of other proteins and discuss its role in folding of this complex Greek key protein. [ABSTRACT FROM AUTHOR]

The solution structure and backbone dynamics of Cu(I) pseudoazurin, a 123 amino acid electron transfer protein from Paracoccus pantotrophus, have been determined using NMR methods. The structure was calculated to high precision, with a backbone RMS deviation for secondary structure elements of 0.35 ± 0.06 +, using 1,498 distance and 55 torsion angle constraints. The protein has a double-wound Greek-key fold with two ±-helices toward its C-terminus, similar to that of its oxidized counterpart determined by X-ray crystallography. Comparison of the Cu(I) solution structure with the X-ray structure of the Cu(II) protein shows only small differences in the positions of some of the secondary structure elements. Order parameters S2, measured for amide nitrogens, indicate that the backbone of the protein is rigid on the picosecond to nanosecond timescale. [ABSTRACT FROM PUBLISHER]

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 β-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., (© 2021 The Protein Society.)

Presents a letter to the editor about the resonance assignment of Cu(I)-pseudoazurin from Alcaligenes faecalis S-6.