Resonance Raman spectra of plastocyanin and pseudoazurin: evidence for conserved cysteine ligand conformations in cupredoxins (blue copper proteins)

New resonance Raman (RR) spectra at 15 K are reported for poplar (Populus nigra) and oleander (Oleander nerium) plastocyanins and for Alcaligenes faecalis pseudoazurin. The spectra are compared with those of other blue copper proteins (cupredoxins). In all cases, nine or more vibrational modes between 330 and 460 cm-1 can be assigned to a coupling of the Cu-S(Cys) stretch with Cys ligand deformations. The fact that these vibrations occur at a relatively constant set of frequencies is testimony to the highly conserved ground-state structure of the Cu-Cys moiety. Shifts of the vibrational modes by 1-3 cm-1 upon deuterium exchange can be correlated with N-H...S hydrogen bonds from the protein backbone to the sulfur of the Cys ligand. There is marked variability in the intensities of these Cys-related vibrations, such that each class of cupredoxin has its own pattern of RR intensities. For example, plastocyanins from poplar, oleander, French bean, and spinach have their most intense feature at approximately 425 cm-1; azurins show greatest intensity at approximately 410 cm-1, stellacyanin and ascorbate oxidase at approximately 385 cm-1, and nitrite reductase at approximately 360 cm-1. These variable intensity patterns are related to differences in the electronic excited-state structures. We propose that they have a basis in the protein environment of the copper-cysteinate chromophore. A further insight into the vibrational spectra is provided by the structures of the six cupredoxins for which crystallographic refinements at high resolution are available (plastocyanins from P. nigra, O. nerium, and Enteromorpha prolifera, pseudoazurin from A. faecalis, azurin from Alcaligenes denitrificans, and cucumber basic blue protein). The average of the Cu-S(Cys) bond lengths is 2.12 +/- 0.05 A. Since the observed range of bond lengths falls within the precision of the determinations, this variation is considered insignificant. The Cys ligand dihedral angles are also highly conserved. Cu-S gamma-C beta-C alpha is always near -170 degrees and S gamma-C beta-C alpha-N near 170 degrees. As a result, the Cu-S gamma bond is coplanar with the Cys side-chain atoms and part of the polypeptide backbone. The coplanarity accounts for the extensive coupling of Cu-S stretching and Cys deformation modes as seen in the RR spectrum. The conservation of this copper-cysteinate conformation in cupredoxins may indicate a favored pathway for electron transfer.     

Crystal structure of the soluble domain of the major anaerobically induced outer membrane protein (AniA) from pathogenic Neisseria: a new class of copper-containing nitrite reductases

The major anaerobically induced outer membrane protein (AniA) from pathogenic Neisseria gonorrhoeae is essential for cell growth under oxygen limiting conditions in the presence of nitrite and is protective against killing by human sera. A phylogenic analysis indicates that AniA is a member of a new class of copper-containing nitrite reductases. Expression of the soluble domain of AniA yields a protein capable of reducing nitrite with specific activity of 160 units/mg, approximately 50 % of that measured for the nitrite reductase from the strong soil denitrifier Alcaligenes faecalis S-6. The crystal structure of the soluble domain of AniA was solved by molecular replacement and sixfold averaging to a resolution of 2.4 A. The nitrite soaked AniA crystal structure refined to 1.95 A reveals a bidentate mode of substrate binding to the type II copper. Despite low sequence identity (approximately 30 %), the core cupredoxin fold of AniA is similar to that found in copper-containing nitrite reductases from soil bacteria. The main structural differences are localized to two attenuated surface loops that map to deletions in the sequence alignment. In soil nitrite reductases, one of these surface loops is positioned near the type I copper site and contributes residues to the docking surface for proteaceous electron donors. In AniA, the attenuation of this loop results in a restructured hydrophobic binding surface that may be required to interact with a lipid anchored azurin. The second attenuated loop is positioned on the opposite side of AniA and may facilitate a more intimate interaction with the lipid membrane. A unique combination of structural effectors surrounding the type I copper site of sAnia contribute to a unusual visible absorption spectra with components observed previously in either green or blue type I copper sites.     

Conformation of pseudoazurin in the 152 kDa electron transfer complex with nitrite reductase determined by paramagnetic NMR

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.     

Bidirectional catalysis by copper-containing nitrite reductase

The copper-containing nitrite reductase from Alcaligenes faecalis S-6 was found to catalyze the oxidation of nitric oxide to nitrite, the reverse of its physiological reaction. Thermodynamic and kinetic constants with the physiological electron donor pseudoazurin were determined for both directions of the catalyzed reaction in the pH range of 6-8. For this, nitric oxide was monitored by a Clark-type electrode, and the redox state of pseudoazurin was measured by optical spectroscopy. The equilibrium constant (K(eq)) depends on the reduction potentials of pseudoazurin and nitrite/nitric oxide, both of which vary with pH. Above pH 6.2 the formation of NiR substrates (nitrite and reduced pseudoazurin) is favored over the products (NO and oxidized pseudoazurin). At pH 8 the K(eq) amounts to 10(3). The results show that dissimilatory nitrite reductases catalyze an unfavorable reaction at physiological pH (pH = 7-8). Consequently, nitrous oxide production by copper-containing nitrite reductases is unlikely to occur in vivo with a native electron donor. With increasing pH, the rate and specificity constant of the forward reaction decrease and become lower than the rate of the reverse reaction. The opposite occurs for the rate of the reverse reaction; thus the catalytic bias for nitrite reduction decreases. At pH 6.0 the k(cat) for nitrite reduction was determined to be 1.5 x 10(3) s(-1), and at pH 8 the rate of the reverse reaction is 125 s(-1).     

Blue copper proteins: a comparative analysis of their molecular interaction properties

Blue copper proteins are type-I copper-containing redox proteins whose role is to shuttle electrons from an electron donor to an electron acceptor in bacteria and plants. A large amount of experimental data is available on blue copper proteins; however, their functional characterization is hindered by the complexity of redox processes in biological systems. We describe here the application of a semiquantitative method based on a comparative analysis of molecular interaction fields to gain insights into the recognition properties of blue copper proteins. Molecular electrostatic and hydrophobic potentials were computed and compared for a set of 33 experimentally-determined structures of proteins from seven blue copper subfamilies, and the results were quantified by means of similarity indices. The analysis provides a classification of the blue copper proteins and shows that (I) comparison of the molecular electrostatic potentials provides useful information complementary to that highlighted by sequence analysis; (2) similarities in recognition properties can be detected for proteins belonging to different subfamilies, such as amicyanins and pseudoazurins, that may be isofunctional proteins; (3) dissimilarities in interaction properties, consistent with experimentally different binding specificities, may be observed between proteins belonging to the same subfamily, such as cyanobacterial and eukaryotic plastocyanins; (4) proteins with low sequence identity, such as azurins and pseudoazurins, can have sufficient similarity to bind to similar electron donors and acceptors while having different binding specificity profiles.     

Two distinct azurins function in the electron-transport chain of the obligate methylotroph Methylomonas J.

Methylomonas J is an obligate methylotroph although it is unable to grow on methane. Like Pseudomonas AM1, it produces two blue copper proteins when growing on methylamine, one of which is the recipient of electrons from the methylamine dehydrogenase. When grown on methanol, only the other blue copper protein is produced. We have determined the amino acid sequences of these blue copper proteins, and show that they are both true azurins. The sequences are clearly homologous to those of the proteins characterized from fluorescent pseudomonads and various species of Alcaligenes, and can be aligned with them and with each other without the need to postulate any internal insertions or deletions in the sequences. The iso-1 azurin, the one produced during both methanol and methylamine growth, shows 59-65% identity with these other azurins, whereas the iso-2 protein shows only 47-53% identity. The proteins show 52% identity with each other. The two functionally equivalent blue copper proteins from Pseudomonas AM1 belong to two sequence classes that are quite distinct from the true azurins. Detailed evidence for the amino acid sequences of the proteins has been deposited as Supplementary Publication SUP 50151 (23 pages) at the British Library Document Supply Centre, Boston Spa, Wetherby, West Yorkshire LS23 7BQ, U.K., from whom copies can be obtained on the terms indicated in Biochem. J. (1989) 257, 5.     

Coordinate synthesis of azurin I and copper nitrite reductase in Alcaligenes xylosoxidans during denitrification

The denitrifying bacterium Alcaligenes xylosoxidans synthesises two azurins (Az), which are termed Az I and Az 2. Both function as effective electron donors to copper nitrite reductase (NiR) in vitro. As a first step towards identifying the physiological relevance of these electron transfer proteins in the denitrification process, the gene (azuA) encoding Az I was characterised and its expression with respect to denitrification determined. We show that the azuA gene from A. xylosoxidans is monocistronic and its expression is increased when cells are grown under denitrifying conditions in the presence of nitrate or nitrite. The expression pattern of azuA was similar, though not identical, to that of the monocistronic nirK gene, which encodes copper NiR, and is in accord with both gene products being synthesised when the bacterium denitrifies. Recombinant Az I was exported to the periplasm of the heterologous host Escherichia coli, was synthesised at very high levels (80 mg purified protein per litre) and was fully loaded with copper. Electron donation from reduced recombinant Az to NiR was indistinguishable from the activity determined with the native protein. Taken together, these findings indicate that in A. xylosoxidans azuA expression is coordinated with denitrification and recombinant Az I is processed and matured in the periplasm of E. coli in the same way it is in A. xylosoxidans.     

Pseudoazurin dramatically enhances the reaction profile of nitrite reduction by Paracoccus pantotrophus cytochrome cd1 and facilitates release of product nitric oxide

Cytochrome cd(1) is a respiratory nitrite reductase found in the periplasm of denitrifying bacteria. When fully reduced Paracoccus pantotrophus cytochrome cd(1) 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) d(1)Fe(II)-NO(+) and cFe(III) d(1)Fe(II)-NO (cd(1)-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 cd(1), 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 cd(1) with nitrite bound to the d(1) heme (i.e. from which NO had dissociated). Pseudoazurin catalyzed this effect even when present at only one-tenth the stoichiometry of cytochrome cd(1). In contrast, redox-inert zinc pseudoazurin did not affect cd(1)-X, indicating a crucial role for electron movement between monomers or individual enzyme dimers rather than simply a protein-protein interaction. Furthermore, formation of cd(1)-X was, remarkably, accelerated by the presence of pseudoazurin, such that it occurred at a rate consistent with cd(1)-X being an intermediate in the catalytic cycle. It is clear that cytochrome cd(1) functions significantly differently in the presence of its two substrates, nitrite and electron donor protein, than in the presence of nitrite alone.     

Redox properties and acid-base equilibria of zucchini mavicyanin

The reduction potential of mavicyanin isolated from zucchini peelings, which is a blue copper protein belonging to the subclass of the phytocyanins, has been determined through direct electrochemistry as a function of temperature and pH. The enthalpy and entropy changes accompanying protein reduction were found to be very similar with those determined previously for other phytocyanins and to differ remarkably from those of azurins and plastocyanins. This finding contributes to further characterize phytocyanins as a distinct cupredoxins family also on thermodynamic grounds and improves our understanding of how the reduction potential of these metal centers in proteins is modulated by coordinative and solvation properties. The E degrees' of mavicyanin is found to be sensitive to two acid-base equilibria at the extremes of pH. One occurs below pH 4, and is related to the protonation and detachment from the Cu(I) center of a histidine ligand. The other, observed above pH 8, causes a remarkable change in the electrostatic potential and/or the field strength around the copper.     

A missing link in cupredoxins: crystal structure of cucumber stellacyanin at 1.6 A resolution.

Stellacyanins are blue (type I) copper glycoproteins that differ from other members of the cupredoxin family in their spectroscopic and electron transfer properties. Until now, stellacyanins have eluded structure determination. Here we report the three-dimensional crystal structure of the 109 amino acid, non-glycosylated copper binding domain of recombinant cucumber stellacyanin refined to 1.6 A resolution. The crystallographic R-value for all 18,488 reflections (sigma > 0) between 50-1.6 A is 0.195. The overall fold is organized in two beta-sheets, both with four beta-stands. Two alpha-helices are found in loop regions between beta-strands. The beta-sheets form a beta-sandwich similar to those found in other cupredoxins, but some features differ from proteins such as plastocyanin and azurin in that the beta-barrel is more flattened, there is an extra N-terminal alpha-helix, and the copper binding site is much more solvent accessible. The presence of a disulfide bond at the copper binding end of the protein confirms that cucumber stellacyanin has a phytocyanin-like fold. The ligands to copper are two histidines, one cysteine, and one glutamine, the latter replacing the methionine typically found in mononuclear blue copper proteins. The Cu-Gln bond is one of the shortest axial ligand bond distances observed to date in structurally characterized type I copper proteins. The characteristic spectroscopic properties and electron transfer reactivity of stellacyanin, which differ significantly from those of other well-characterized cupredoxins, can be explained by its more exposed copper site, its distinctive amino acid ligand composition, and its nearly tetrahedral ligand geometry. Surface features on the cucumber stellacyanin molecule that could be involved in interactions with putative redox partners are discussed.     

Role of ligand substitution on long-range electron transfer in azurins.

Azurin contains two potential redox sites, a copper centre and, at the opposite end of the molecule, a cystine disulfide (RSSR). Intramolecular electron transfer between a pulse radiolytically produced RSSR- radical anion and the blue Cu(II) ion was studied in a series of azurins in which single-site mutations were introduced into the copper ligand sphere. In the Met121His mutant, the rate constant for intramolecular electron transfer is half that of the corresponding wild-type azurin. In the His46Gly and His117Gly mutants, a water molecule is co-ordinated to the copper ion when no external ligands are added. Both these mutants also exhibit slower intramolecular electron transfer than the corresponding wild-type azurin. However, for the His117Gly mutant in the presence of excess imidazole, an azurin-imidazole complex is formed and the intramolecular electron-transfer rate increases considerably, becoming threefold faster than that observed in the native protein. Activation parameters for all these electron-transfer processes were determined and combined with data from earlier studies on intramolecular electron transfer in wild-type and single-site-mutated azurins. A linear relationship between activation enthalpy and activation entropy was observed. These results are discussed in terms of reorganization energies, driving force and possible electron-transfer pathways.     

The structure of the Met144Leu mutant of copper nitrite reductase from <Emphasis Type="Italic">Alcaligenes xylosoxidans</Emphasis> provides the first glimpse of a protein–protein complex with azurin II

Cu-containing nitrite reductases (NiRs) perform the reduction of nitrite to NO via an ordered mechanism in which the delivery of a proton and an electron to the catalytic type 2 Cu site is highly orchestrated. Electron transfer from a redox partner protein, azurin or pseudoazurin, to the type 1 Cu site is assumed to occur through the formation of a protein-protein complex. We report here a new crystal form in space group P2(1)2(1)2(1) of the Met144Leu mutant of NiR from Alcaligenes xylosoxidans (AxNiR), revealing a head-to-head packing motif involving residues around the hydrophobic patch of domain 1. Superposition of the structure of azurin II with that of domain 1 of one of the Met144Leu molecules provides the first glimpse of an azurin II-NiR protein-protein complex. Mutations of two of the residues of AxNiR, Trp138His (Barrett et al. in Biochemistry 43:16311-16319, 2004) and Met87Leu, highlighted in the AxNiR-azurin complex, results in substantially decreased activity when azurin is used as the electron donor instead of methyl viologen, providing direct evidence for the importance of this region for complex formation.     

Metal-ligand interactions in perturbed blue copper sites: a paramagnetic 1H NMR study of Co(II)-pseudoazurin

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 (1)H NMR spectra of Co(II)-substituted pseudoazurin from Achromobacter cycloclastes. The (1)H 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 Sgamma(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)-S(cys) bond is sensibly smaller than the Cu(II)-S(cys). Notwithstanding, NMR data on Co(II)-substituted blue copper proteins can be safely extrapolated to native Cu(II) proteins.     

Two c-type cytochromes, NirM and NirC, encoded in the nir gene cluster of Pseudomonas aeruginosa act as electron donors for nitrite reductase.

Three c-type cytochromes, NirM, NirC, and NirN, are encoded in the nirSMCFDLGHJEN gene cluster for cytochrome cd(1)-type nitrite reductase (NIR) of Pseudomonas aeruginosa. nirS is the structural gene for NIR. NirM (cytochrome c(551)) is reported to be a physiological electron donor for nitrite reductase. The respective functions of NirC and NirN have remained unclear. In this study, we produced recombinant NirC and NirN in P. aeruginosa, and purified them from the periplasmic fraction. N-terminal amino acid sequences of the purified proteins showed that the N-terminal 31 and 18 residues of NirC and NirN precursors were cleaved, respectively, indicating that cleaved peptides act as signals for membrane translocation. In addition, the ability of NirC for electron donation to nitrite reductase was investigated. NirC, as well as NirM, was able to mediate the electron donation from the membrane electron pathway to NIR, suggesting that the structural gene for NIR is followed by the genes for two electron donors for NIR.     

Structural comparison of cupredoxin domains: domain recycling to construct proteins with novel functions.

The three-dimensional structures of the copper-containing enzymes ascorbate oxidase, ceruloplasmin, and nitrite reductase, comprised of multiple domains with a cupredoxin fold, are consistent with having evolved from a common ancestor. The presence or absence of copper sites has complicated ascertaining the structural and evolutionary relationship among these and related proteins. Simultaneous structural superposition of the enzyme domains and their known cupredoxin relatives shows clearly that there are at least six cupredoxin classes, and that the evolution of the conserved core of these domains is independent of the presence or absence of copper sites. Relationships among the variable loops in these structures show that the two-domain ancestor of the blue oxidases contained a trinuclear-copper interface but could not have functioned in a monomeric state. Comparison of the sequence of the copper-containing, iron-regulating protein. Ferrous transport (Fet3) from yeast to the structurally defined core and loop residues of the cupredoxins suggests specific residues that could be involved in the ferroxidase activity of Fet3.     

Cytochrome cd1, reductive activation and kinetic analysis of a multifunctional respiratory enzyme.

Paracoccus pantotrophus cytochrome cd(1) is an enzyme of bacterial respiration, capable of using nitrite in vivo and also hydroxylamine and oxygen in vitro as electron acceptors. We present a comprehensive analysis of the steady state kinetic properties of the enzyme with each electron acceptor and three electron donors, pseudoazurin and cytochrome c(550), both physiological, and the non-physiological horse heart cytochrome c. At pH 5.8, optimal for nitrite reduction, the enzyme has a turnover number up to 121 s(-1) per d(1) heme, significantly higher than previously observed for any cytochrome cd(1). Pre-activation of the enzyme via reduction is necessary to establish full catalytic competence with any of the electron donor proteins. There is no significant kinetic distinction between the alternative physiological electron donors in any respect, providing support for the concept of pseudospecificity, in which proteins with substantially different tertiary structures can transfer electrons to the same acceptor. A low level hydroxylamine disproportionase activity that may be an intrinsic property of cytochromes c is also reported. Important implications for the enzymology of P. pantotrophus cytochrome cd(1) are discussed and proposals are made about the mechanism of reduction of nitrite, based on new observations placed in the context of recent rapid reaction studies.     

The blue copper-containing nitrite reductase from Alcaligenes xylosoxidans: cloning of the nirA gene and characterization of the recombinant enzyme

The nirA gene encoding the blue dissimilatory nitrite reductase from Alcaligenes xylosoxidans has been cloned and sequenced. To our knowledge, this is the first report of the characterization of a gene encoding a blue copper-containing nitrite reductase. The deduced amino acid sequence exhibits a high degree of similarity to other copper-containing nitrite reductases from various bacterial sources. The full-length protein included a 24-amino-acid leader peptide. The nirA gene was overexpressed in Escherichia coli and was shown to be exported to the periplasm. Purification was achieved in a single step, and analysis of the recombinant Nir enzyme revealed that cleavage of the signal peptide occurred at a position identical to that for the native enzyme isolated from A. xylosoxidans. The recombinant Nir isolated directly was blue and trimeric and, on the basis of electron paramagnetic resonance spectroscopy and metal analysis, possessed only type 1 copper centers. This type 2-depleted enzyme preparation also had a low nitrite reductase enzyme activity. Incubation of the periplasmic fraction with copper sulfate prior to purification resulted in the isolation of an enzyme with a full complement of type 1 and type 2 copper centers and a high specific activity. The kinetic properties of the recombinant enzyme were indistinguishable from those of the native nitrite reductase isolated from A. xylosoxidans. This rapid isolation procedure will greatly facilitate genetic and biochemical characterization of both wild-type and mutant derivatives of this protein.     

Transient kinetics of reduction of blue copper proteins by free flavin and flavodoxin semiquinones

Rate constants have been determined for the electron-transfer reactions between reduced free flavins and flavodoxin semiquinone and several blue copper proteins. Correlations between these values and redox potentials demonstrate that spinach plastocyanin, Pseudomonas aeruginosa azurin, Alcaligenes sp. azurin, and Alcaligenes sp. nitrite reductase have the same intrinsic reactivities toward free flavins, whereas stellacyanin is more reactive (3.3 times) and laccase considerably less reactive (approximately 12 times). Electrostatic interactions between the negatively charged flavin mononucleotide (FMN) and the copper proteins show that the interaction site charges for laccase and nitrite reductase are opposite in sign to the net protein charge and that the signs and magnitudes of the charges are consistent with the known three-dimensional structures for plastocyanin and the azurins and with amino acid sequence homologies for stellacyanin. The results demonstrate that the apparent interaction site charge with flavodoxin is larger than that with FMN for plastocyanin, nitrite reductase, and stellacyanin but smaller for Pseudomonas azurin. This is interpreted in terms of a larger interaction domain for the flavodoxin reaction, which allows charged groups more distant from the actual electron-transfer site to become involved. The intrinsic reactivities of plastocyanin and azurin toward flavodoxin are the same, as was the case with FMN, but both stellacyanin and nitrite reductase are considerably less reactive than expected (approximately 2 orders of magnitude). This result suggests the involvement of steric factors with these latter two proteins which discriminate against large reactants such as flavodoxin.