The CO dehydrogenase accessory protein CooT is a novel nickel-binding protein.

In Rhodospirillum rubrum, maturation of Carbon Monoxide Dehydrogenase (CODH) requires three accessory proteins, CooC, CooT and CooJ, dedicated to nickel insertion into the active site, which is constituted by a distorted [NiFe3S4] cubane coordinated with a mononuclear Fe site. CooC is an ATPase proposed to provide the energy required for the maturation process, while CooJ is described as a metallochaperone with 16 histidines and 2 cysteines at the C-terminus, likely involved in metal binding and/or storage. Prior to the present study, no information was available on CooT at the molecular level. Here, the X-ray structure of RrCooT was obtained, which revealed that this protein is a homodimer featuring a fold that resembles an Sm-like domain, suggesting a role in RNA metabolism that was however not supported by experimental observations. Biochemical and biophysical evidence based on circular dichroism spectroscopy, light scattering, isothermal titration calorimetry and site-directed mutagenesis showed that RrCooT specifically binds a single Ni(ii) per dimer, with a dissociation constant of 9 nM, through the pair of Cys2, highly conserved residues, located at the dimer interface. Despite its role in the activation of RrCODH in vivo, CooT was thought to be a unique protein, found only in R. rubrum, with an unclear function. In this study, we extended the biological impact of CooT, establishing that this protein is a member of a novel Ni(ii)-binding protein family with 111 homologues, linked to anaerobic metabolism in bacteria and archaea, and in most cases to the presence of CODH.

Inactivation of urease by 1,4-benzoquinone: chemistry at the protein surface.

The high activity of urease, a Ni(ii) enzyme, has several adverse effects on human health and agriculture, and its modulation needs the use of inhibitors. 1,4-Benzoquinone (BQ) irreversibly inactivates Sporosarcina pasteurii urease (SPU), with first order kinetics for both the inhibitor and the enzyme. This reaction is stoichiometrically quenched in the presence of sulphite. The 2.07 Å crystal structure of SPU bound to BQ shows the presence of a 1,4-hydroquinone moiety covalently bound to the thiol group of αCys322, a key residue found on the mobile flap regulating the substrate access to the active site. The 1.75 Å crystal structure obtained when sulphite is added to a solution of SPU previously incubated with BQ shows the presence of a 2,5-dihydroxy-benzenesulphonate moiety bound to the αCys322 thiol group. These data reveal how the active site cysteine reacts with a prototypical BQ moiety, found in a large number of natural substances potentially suitable to control the urease activity.

Structure-based rationalization of urease inhibition by phosphate: novel insights into the enzyme mechanism.

The structure of Bacillus pasteurii urease (BPU) inhibited with phosphate was solved and refined using synchrotron X-ray diffraction data from a vitrified crystal (1.85 A resolution, 99.3% completeness, data redundancy 4.6, R-factor 17.3%, PDB code 6UBP). A distance of 3.5 A separates the two Ni ions in the active site. The binding mode of the inhibitor involves the formation of four coordination bonds with the two Ni ions: one phosphate oxygen atom symmetrically bridges the two metal ions (1.9-2.0 A), while two of the remaining phosphate oxygen atoms bind to the Ni atoms at 2.4 A. The fourth phosphate oxygen is directed into the active site channel. Analysis of the H-bonding network around the bound inhibitor indicates that phosphate is bound as the H2PO4- anion, and that an additional proton is present on the Odelta2 atom of Asp(alpha363), an active site residue involved in Ni coordination through Odelta1. The flexible flap flanking the active site cavity is in the open conformation. Analysis of the complex reveals why phosphate is a relatively weak inhibitor and why sulfate does not bind to the nickels in the active site. The implications of the results for the understanding of the urease catalytic mechanism are reviewed. A novel alternative for the proton donor is presented.

Structural basis for Ni(2+) transport and assembly of the urease active site by the metallochaperone UreE from Bacillus pasteurii.

Bacillus pasteurii UreE (BpUreE) is a putative chaperone assisting the insertion of Ni(2+) ions in the active site of urease. The x-ray structure of the protein has been determined for two crystal forms, at 1.7 and 1.85 A resolution, using SIRAS phases derived from a Hg(2+)-derivative. BpUreE is composed of distinct N- and C-terminal domains, connected by a short flexible linker. The structure reveals the topology of an elongated homodimer, formed by interaction of the two C-terminal domains through hydrophobic interactions. A single Zn(2+) ion bound to four conserved His-100 residues, one from each monomer, connects two dimers resulting in a tetrameric BpUreE known to be formed in concentrated solutions. The Zn(2+) ion can be replaced by Ni(2+) as shown by anomalous difference maps obtained on a crystal of BpUreE soaked in a solution containing NiCl(2). A large hydrophobic patch surrounding the metal ion site is surface-exposed in the biologically relevant dimer. The BpUreE structure represents the first for this class of proteins and suggests a possible role for UreE in the urease nickel-center assembly.

Backbone dynamics of plastocyanin in both oxidation states. Solution structure of the reduced form and comparison with the oxidized state.

A model-free analysis based on (15)N R(1), (15)N R(2), and (15)N-(1)H nuclear Overhauser effects was performed on reduced (diamagnetic) and oxidized (paramagnetic) forms of plastocyanin from Synechocystis sp. PCC6803. The protein backbone is rigid, displaying a small degree of mobility in the sub-nanosecond time scale. The loops surrounding the copper ion, involved in physiological electron transfer, feature a higher extent of flexibility in the longer time scale in both redox states, as measured from D(2)O exchange of amide protons and from NH-H(2)O saturation transfer experiments. In contrast to the situation for other electron transfer proteins, no significant difference in the dynamic properties is found between the two redox forms. A solution structure was also determined for the reduced plastocyanin and compared with the solution structure of the oxidized form in order to assess possible structural changes related to the copper ion redox state. Within the attained resolution, the structure of the reduced plastocyanin is indistinguishable from that of the oxidized form, even though small chemical shift differences are observed. The present characterization provides information on both the structural and dynamic behavior of blue copper proteins in solution that is useful to understand further the role(s) of protein dynamics in electron transfer processes.

The first solution structure of a paramagnetic copper(II) protein: the case of oxidized plastocyanin from the cyanobacterium Synechocystis PCC6803.

The NMR solution structure of oxidized plastocyanin from the cyanobacterium Synechocystis PCC6803 is here reported. The protein contains paramagnetic copper(II), whose electronic relaxation times are quite unfavorable for NMR solution studies. The structure has been solved on the basis of 1041 meaningful NOESY cross-peaks, 18 1D NOEs, 26 T(1) values, 96 dihedral angle constraints, and 18 H-bonds. The detection of broad hyperfine-shifted signals and their full assignment allowed the identification of the copper(II) ligands and the determination of the Cu-S-C-H dihedral angle for the coordinated cysteine. The global root-mean-square deviation from the mean structure for the solution structure family is 0.72 +/- 0.14 and 1.16 +/- 0.17 A for backbone and heavy atoms, respectively. The structure is overall quite satisfactory and represents a breakthrough, in that it includes paramagnetic copper proteins among the metalloproteins for which solution structures can be afforded. The comparison with the available X-ray structure of a triple mutant is also performed.

Structure-based computational study of the catalytic and inhibition mechanisms of urease.

The viability of different mechanisms of catalysis and inhibition of the nickel-containing enzyme urease was explored using the available high-resolution structures of the enzyme isolated from Bacillus pasteurii in the native form and inhibited with several substrates. The structures and charge distribution of urea, its catalytic transition state, and three enzyme inhibitors were calculated using ab initio and density functional theory methods. The DOCK program suite was employed to determine families of structures of urease complexes characterized by docking energy scores indicative of their relative stability according to steric and electrostatic criteria. Adjustment of the parameters used by DOCK, in order to account for the presence of the metal ion in the active site, resulted in the calculation of best energy structures for the nickel-bound inhibitors beta-mercaptoethanol, acetohydroxamic acid, and diamidophosphoric acid. These calculated structures are in good agreement with the experimentally determined structures, and provide hints on the reactivity and mobility of the inhibitors in the active site. The same docking protocol was applied to the substrate urea and its catalytic transition state, in order to shed light onto the possible catalytic steps occurring at the binuclear nickel active site. These calculations suggest that the most viable pathway for urea hydrolysis involve a nucleophilic attack by the bridging, and not the terminal, nickel-bound hydroxide onto a urea molecule, with active site residues playing important roles in orienting and activating the substrate, and stabilizing the catalytic transition state.

Crystal structure of oxidized Bacillus pasteurii cytochrome c553 at 0.97-A resolution.

This article reports the first X-ray structure of the soluble form of a c-type cytochrome isolated from a Gram-positive bacterium. Bacillus pasteurii cytochrome c(553), characterized by a low reduction potential and by a low sequence homology with cytochromes from Gram-negative bacteria or eukaryotes, is a useful case study for understanding the structure-function relationships for this class of electron-transfer proteins. Diffraction data on a single crystal of cytochrome c(553) were obtained using synchrotron radiation at 100 K. The structure was determined at 0.97-A resolution using ab initio phasing and independently at 1.70 A in an MAD experiment. In both experiments, the structure solution exploited the presence of a single Fe atom as anomalous scatterer in the protein. For the 0.97-A data, the phasing was based on a single data set. This is the most precise structure of a heme protein to date. The crystallized cytochrome c(553) contains only 71 of the 92 residues expected from the intact protein sequence, lacking the first 21 amino acids at the N-terminus. This feature is consistent with previous evidence that this tail, responsible for anchoring the protein to the cytoplasm membrane, is easily cleaved off during the purification procedure. The heme prosthetic group in B. pasteurii cytochrome c(553) is surrounded by three alpha-helices in a compact arrangement. The largely exposed c-type heme group features a His-Met axial coordination of the Fe(III) ion. The protein is characterized by a very asymmetric charge distribution, with the exposed heme edge located on a surface patch devoid of net charges. A structural search of a representative set of protein structures reveals that B. pasteurii cytochrome c(553) is most similar to Pseudomonas cytochromes c(551), followed by cytochromes c(6), Desulfovibrio cytochrome c(553), cytochromes c(552) from thermophiles, and cytochromes c from eukaryotes. Notwithstanding a low sequence homology, a structure-based alignment of these cytochromes shows conservation of three helical regions, with different additional secondary structure motifs characterizing each protein. In B. pasteurii cytochrome c(553), these motifs are represented by the shortest interhelix connecting fragments observed for this group of proteins. The possible relationships between heme solvent accessibility and the electrochemical reduction potential are discussed.

The complex of Bacillus pasteurii urease with acetohydroxamate anion from X-ray data at 1.55 A resolution.

The structure of Bacillus pasteurii urease inhibited with acetohydroxamic acid was solved and refined anisotropically using synchrotron X-ray cryogenic diffraction data (1.55 A resolution, 99.5% completeness, data redundancy = 26, R-factor = 15.1%, PDB code 4UBP). The two Ni ions in the active site are separated by a distance of 3.53 A. The structure clearly shows the binding mode of the inhibitor anion, symmetrically bridging the two Ni ions in the active site through the hydroxamate oxygen and chelating one Ni ion through the carbonyl oxygen. The flexible flap flanking the active site cavity is in the open conformation. The possible implications of the results on structure-based molecular design of new urease inhibitors are discussed.

Cytochrome c-553 from the alkalophilic bacterium Bacillus pasteurii has the primary structure characteristics of a lipoprotein.

The complete sequence of Bacillus pasteurii cytochrome c-553 was determined by standard methods of Edman degradation of overlapping peptides combined with mass spectrometry. The protein contains 92 residues and a single heme-binding site. It is most similar to Bacillus licheniformis, Bacillus PS3, and Bacillus subtilis cytochromes c-551, which are lipoproteins that are partially solubilized through proteolytic cleavage of the N-terminal diacyl-glyceryl-cysteine membrane anchor. The high yield of the B. pasteurii cytochrome c-553, together with evidence that shorter forms of the cytochrome occur in the mixture of otherwise pure protein, suggests that the membrane anchor is very susceptible to proteolysis and that the soluble form of the cytochrome is therefore released from the membrane upon cell breakage. A sequence-based calculation of the protein secondary structure suggests the presence of a typical cytochrome helical fold with a random-coil N-terminus tail.

A new proposal for urease mechanism based on the crystal structures of the native and inhibited enzyme from Bacillus pasteurii: why urea hydrolysis costs two nickels.

BACKGROUND: Urease catalyzes the hydrolysis of urea, the final step of organic nitrogen mineralization, using a bimetallic nickel centre. The role of the active site metal ions and amino acid residues has not been elucidated to date. Many pathologies are associated with the activity of ureolytic bacteria, and the efficiency of soil nitrogen fertilization with urea is severely decreased by urease activity. Therefore, the development of urease inhibitors would lead to a reduction of environmental pollution, to enhanced efficiency of nitrogen uptake by plants, and to improved therapeutic strategies for treatment of infections due to ureolytic bacteria. Structure-based design of urease inhibitors would require knowledge of the enzyme mechanism at the molecular level. RESULTS: The structures of native and inhibited urease from Bacillus pasteurii have been determined at a resolution of 2.0 A by synchrotron X-ray cryogenic crystallography. In the native enzyme, the coordination sphere of each of the two nickel ions is completed by a water molecule and a bridging hydroxide. A fourth water molecule completes a tetrahedral cluster of solvent molecules. The enzyme crystallized in the presence of phenylphosphorodiamidate contains the tetrahedral transition-state analogue diamidophosphoric acid, bound to the two nickel ions in an unprecedented mode. Comparison of the native and inhibited structures reveals two distinct conformations of the flap lining the active-site cavity. CONCLUSIONS: The mode of binding of the inhibitor, and a comparison between the native and inhibited urease structures, indicate a novel mechanism for enzymatic urea hydrolysis which reconciles the available structural and biochemical data.

On the role of high-potential iron-sulfur proteins and cytochromes in the respiratory chain of two facultative phototrophs

The capability of high potential iron-sulfur proteins (HiPIPs) and soluble cytochromes to shuttle electrons between the bc1 complex and the terminal oxidase in aerobically grown cells of Rhodoferax fermentans and Rhodospirillum salinarum, two facultative phototrophs, was evaluated. In Rs. salinarum, HiPIP and a c-type cytochrome (alpha-band at 550 nm, Em,7=+290 mV) are both involved in the electron transfer step from the bc1 complex to the terminal oxidase. Kinetic studies indicate that cytochrome c550 is more efficient than HiPIP in oxidizing the bc1 complex, and that HiPIP is a more efficient reductant of the terminal oxidase as compared to cytochrome c550. Rs. salinarum cells contain an additional c-type cytochrome (asymmetric alpha-band at 556 nm, Em,7=+180 mV) which is able to reduce the terminal oxidase, but unable to oxidize the bc1 complex. c-type cytochromes could not be isolated from Rf. fermentans, in which HiPIP, the most abundant soluble electron carrier, is reduced by the bc1 complex (zero-order kinetics) and oxidized by the terminal oxidase (first-order kinetics), respectively. These data, taken together, indicate for the first time that HiPIPs play a significant role in bacterial respiratory electron transfer.

Crystallization and preliminary X-ray diffraction analysis of cytochrome c' from Rubrivivax gelatinosus at 1.3 A resolution.

Cytochrome c' from the purple non-sulfur phototrophic bacterium Rubrivivax gelatinosus has been crystallized by vapour diffusion at pH 5, 6.3 and 8, in sodium acetate, sodium citrate, and Tris-HCl buffers, respectively. Crystals grown at pH 5 and 6.3 diffract, respectively, to 2.0 A (298 K) and 1.4 A (100 K) using synchrotron radiation. Data up to 1.3 A resolution with 99.8% completeness were collected at 100 K on a crystal grown at pH 8. The space group is P3121 or P3221, and the unit-cell parameters are a = b = 69.63, c = 123.63 A.

Crystallization and preliminary high-resolution X-ray diffraction analysis of native and beta-mercaptoethanol-inhibited urease from Bacillus pasteurii.

Hexagonal crystals of urease from Bacillus pasteurii have been obtained by vapour diffusion at 293 K in 20 mM Tris-HCl, neutral pH, containing 50 mM Na2SO3. Isomorphous crystals of urease inhibited with beta-mercaptoethanol were also obtained by including 4 mM of the inhibitor in the enzyme solution. Crystals of the native and inhibited enzyme diffract respectively to 2.00 A (96.7% completeness) and to 1.65 A (98.7% completeness) using synchrotron X-ray cryogenic (100 K) conditions. The space group is P6322 for both forms, and the unit-cell parameters are a = b = 131.36, c = 189. 76 A for native urease and a = b = 131.34, c = 190.01 A for inhibited urease. Under the same conditions, single crystals of B. pasteurii urease inhibited with acetohydroxamic acid, cisteamine, and phenylphosphorodiamidate were also obtained.

Crystals of cytochrome c-553 from Bacillus pasteurii show diffraction to 0.97 A resolution.

We report here the purification and characterization of a c-type cytochrome present in the soluble fraction of the gram-positive, alkaliphilic, and highly ureolytic soil bacterium Bacillus pasteurii. The cytochrome is acidic (pI = 3.3), has a molecular mass of 9.5 kDa, and appears to dimerize in 150 mM ionic strength solution. The electronic spectrum is typical of a low-spin hexa-coordinated heme iron. Crystals of the protein in the oxidized state were grown by vapor diffusion at pH 5, by using 3.2 M ammonium sulfate as precipitant. Diffraction data at ultrahigh resolution (0.97 A) and completeness (99.9%) have been collected under cryogenic conditions, by using synchrotron radiation. The crystals belong to the orthorhombic space group P2(1)2(1)2(1), with cell constants a = 37.14, b = 39.42, c = 44.02 A, and one protein monomer per asymmetric unit. Attempts to solve the crystal structure by ab initio methods are in progress.

The primary structure of Rhodoferax fermentans high-potential iron-sulfur protein, an electron donor to the photosynthetic reaction center.

The complete amino acid sequence of Rhodoferax fermentans high-potential iron-sulfur protein (Hipip), which is known to be an efficient electron donor to the photosynthetic reaction center, has been determined using both N-terminal and C-terminal analyses. The sequence contains 75 residues, with 11 positive charges, 10 negative charges, and one histidine residue. The molecular mass of apo-Hipip, determined by electrospray ionization mass spectrometry, is 7849.64 Da. Multiple sequence alignment, based both on primary and tertiary structure information, reveals conservation of Tyr19 and Gly75 (Chromatium vinosum numbering) in addition to the four [Fe4S4]-bound cysteines. The Hipip from Rf. fermentans is most similar (57% similarity) to the Hipip from Rubrivivax gelatinosus, a photosynthetic bacterium belonging to the beta-1 subgroup of the proteobacteria.

Kinetics of photo-induced electron transfer from high-potential iron-sulfur protein to the photosynthetic reaction center of the purple phototroph Rhodoferax fermentans.

The kinetics of photo-induced electrontransfer from high-potential iron-sulfur protein (HiPIP) to the photosynthetic reaction center (RC) of the purple phototroph Rhodoferarfermentans were studied. The rapid photooxidation of heme c-556 belonging to RC is followed, in the presence of HiPIP, by a slower reduction having a second-order rate constant of 4.8 x 10(7) M(-1) x s(-1). The limiting value of kobs at high HiPIP concentration is 95 s(-1). The amplitude of this slow process decreases with increasing HiPIP concentration. The amplitude of a faster phase, observed at 556 and 425 nm and involving heme c-556 reduction, increases proportionately. The rate constant of this fast phase, determined at 425 and 556 nm, is approximately 3 x 10(5) s(-1). This value is not dependent on HiPIP concentration, indicating that it is related to a first-order process. These observations are interpreted as evidence for the formation of a HiPIP-RC complex prior to the excitation flash, having a dissociation constant of -2.5 microM. The fast phase is absent at high ionic strength, indicating that the complex involves mainly electrostatic interactions. The ionic strength dependence of kobs for the slow phase yields a second-order rate constant at infinite ionic strength of 5.4 x 10(6) M(-1) x s(-1) and an electrostatic interaction energy of -2.1 kcal/mol (1 cal = 4.184 J). We conclude that Rhodoferar fermentans HiPIP is a very effective electron donor to the photosynthetic RC.

X-ray absorption spectroscopy study of native and phenylphosphorodiamidate-inhibited Bacillus pasteurii urease.

X-ray absorption spectroscopy (XAS) has been applied to urease from Bacillus pasteurii, a highly ureolytic soil bacterium, with the aim of elucidating the structural details of the nickel-containing active site. The results indicate the presence of octahedrally coordinated Ni2+, in a sphere of six N/O donors at an average distance of 0.203 nm. An average of two histidine residues are bound to nickel. The experimental evidence suggests direct binding of the urease inhibitor phenylphosphorodiamidate to Ni2+. These spectroscopic results are in agreement with previous findings on both plant and microbial ureases, but differ in some respect from the results obtained by X-ray crystallography analysis of Klebsiella aerogenes urease.

1H NMR of high-potential iron-sulfur protein from the purple non-sulfurbacterium Rhodoferax fermentans.

Oxidized and reduced forms of high-potential iron-sulfur protein (HiPIP) from the purple non-sulfur photosynthetic bacterium Rhodoferax fermentans have been characterized using 1H-NMR spectroscopy. Pairwise and sequence-specific assignments of hyperfine-shifted 1H-NMR signals to protons of cysteine residues bound to the [4Fe-4S]3+/2+ cluster have been performed using one-dimensional NOE and exchange spectroscopy experiments. 1H-NMR hyperfine shifts and relaxation rates of cluster-bound Cys beta-CH2 protons indicate that in the [4Fe-4S]3+ cluster one iron ion can be formally described as Fe(III), while electron density corresponding to one electron is unevenly delocalized onto the remaining three iron ions. This delocalization is effected by means of two different electronic distributions interconverting rapidly on the NMR time scale. The mechanism of paramagnetic proton relaxation, studied by analyzing longitudinal relaxation rates of Cys beta-CH2 protons in HiPIPs from six different sources as a function of the Fe-S-C beta-C alpha dihedral angle, indicate that the major contribution is due to a dipolar metal-centered mechanism, with a non-negligible contribution from a ligand-centered dipolar mechanism which involves the 3p orbital of the Cys sulfur atom. A semi-quantitative tool for extracting structural information from relaxation time measurements is proposed.

Isolation, characterization, and functional role of the high-potential iron-sulfur protein (HiPIP) from Rhodoferax fermentans.

A new high-potential iron-sulfur protein (HiPIP) has been isolated and purified to homogeneity from the soluble fraction obtained from light-grown cells of the facultative photoheterotrophic bacterium Rhodoferax fermentans. The new protein was identified as a HiPIP by virtue of its molecular properties such as the molecular mass (M(r) = 8.7 kDa), the Fe/protein ratio (3.8 +/- 0.2), the reduction potential (Em,7 = +351 mV), the electronic spectrum of the reduced and the oxidized protein, and the EPR spectrum of the oxidized protein. These molecular properties lie in the range observed for HiPIPs from other sources and, in particular, the iron content is consistent with the presence of one [Fe4S4] cubane cluster per molecule. The isoelectric pH values of the two redox forms are consistent with a basic protein. Kinetic studies of HiPIP oxidation, performed by monitoring the absorbance changes induced upon light excitation of the photosynthetic reaction center, give direct evidence of the role of the HiPIP in the photosynthetic electron transfer chain of Rf. fermentans.