PubMed Health⌕ Search

Biomedical subjects

S S Hasnain

Publications and source records attributed to S S Hasnain.

At least 37 records · Page 2Linked to original sources
PubMed1999

Structural and kinetic evidence for an ordered mechanism of copper nitrite reductase.

The crystallographic structures of several copper-containing nitrite reductases are now available. Despite this wealth of structural data, no definitive information is available as to whether the reaction proceeds by an ordered mechanism where nitrite binds to the oxidised type 2 site, followed by an internal electron transfer from the type 1 Cu, or whether binding occurs to the reduced type 2 Cu centre, or a random mechanism operates. We present here the first structural information on both types of Cu centres for the reduced form of NiR from Alcaligenes xylosoxidans (AxNiR) using X-ray absorption spectroscopy. The reduced type 2 Cu site EXAFS shows striking similarity to the EXAFS data for reduced bovine superoxide dismutase (Cu2Zn2 SOD), providing strong evidence for the loss of the water molecule from the catalytic Cu site in NiR on reduction resulting in a tri-coordinate Cu site similar to that in Cu2Zn2 SOD. The reduced type 2 Cu site of AxNiR is shown to be unable to bind inhibitory ligands such as azide, and to react very sluggishly with nitrite leading to only a slow re-oxidation of the the type 1 centre. These observations provide strong evidence that turnover of AxNiR proceeds by an ordered mechanism in which nitrite binds to the oxidised type 2 Cu centres before electron transfer from the reduced type 1 centre occurs. We propose that the two links between the Cu sites of AxNiR, namely His129-Cys130 and His89-Asp92-His94 are utilised for electron transfer and for communicating the status of the type 2 Cu site, respectively. Nitrite binding at type 2 Cu is sensed by the proton abstracting group Asp92 and the type 2 Cu ligand His94, and relayed to the type 1 Cu site via His89 thus triggering an internal electron transfer. The similarity of the type 2 Cu NiR catalytic site to the reduced Cu site of SOD is examined in some detail together with the biochemical evidence for the SOD activity of AxNiR.

Absorption↗
PubMed1999

Crystallographic structures of bovine copper-zinc superoxide dismutase reveal asymmetry in two subunits: functionally important three and five coordinate copper sites captured in the same crystal.

A key feature of the generally accepted catalytic mechanism of CuZn superoxide dismutase (CuZnSOD) is the breakage of the imidazolate bridge between copper and zinc and the loss of a coordinated water molecule from copper on reduction from Cu(II) to Cu(I). Crystal structures exist for the enzyme from a number of sources in the oxidised, five coordinate copper form. For the reduced form two structures from different sources have been determined only recently but provide contradictory results. We present crystal structures of bovine CuZnSOD (BSOD) in two different space groups. The structure of the P212121 form (pBSOD), at 1.65 A resolution clearly shows one subunit with Cu in the five coordinate, oxidised form, and the other with Cu in the three coordinate form expected for the reduced state. This mixed state of pBSOD is confirmed by XANES data of these crystals. The pBSOD structure has thus captured each subunit in one of the two oxidation state conformations and thus provides direct crystallographic evidence for the superoxide dismutase mechanism involving the breakage of the imidazole bridge between Cu and Zn. A shift in the position of copper in subunit A poises the catalytic centre to undergo the first stage of catalysis via dissociation of Cu from His61 with a concomittant movement of the coordinated water molecule towards His61, which rotates by approximately 20 degrees, enabling it to form a hydrogen bond to the water molecule. The Cu-Zn separation in the reduced site is increased by approximately 0.5 A. In contrast the 2.3 A resolution structure in space group C2221 (cBSOD) shows both of the Cu atoms to be in the five coordinate, oxidised form but in this space group the whole of subunit A is significantly more disordered than subunit B. An examination of published structures of "oxidised" SODs, shows a trend towards longer Cu-Zn and Cu-His61 separations in subunit A, which together with the structures reported here indicate a potential functional asymmetry between the subunits of CuZnSODs. We also suggest that the increased separation between Cu and Zn is a precursor to breakage of His61.

Animals↗
PubMed1999

Ab initio phasing using molecular envelope from solution X-ray scattering.

Solving the phase problem is the crucial and quite often the most difficult and time-consuming step in crystallographic structure determination. The traditional methods of isomorphous replacement (MIR or SIR) and molecular replacement require the availability of an isomorphous heavy-atom derivative or the structure of a homologous protein, respectively. Here, a method is presented which utilizes the low-resolution molecular shape determined from solution X-ray scattering data for the molecular search. The molecular shape of a protein is an important structural property and can be determined directly by the small-angle scattering technique. The idea of locating this molecular shape in the crystallographic unit cell has been tested with experimental diffraction data from nitrite reductase (NiR). The conventional Patterson search proved to be unsuccessful, as the intra-envelope vectors are uniformly distributed and do not match those of intra-molecular (atom-to-atom) vectors. A direct real-space search for orientation and translation was then performed. A self-rotation function using 2.8 A crystallographic data yielded the polar angles of the non-crystallographic threefold axis. Knowledge of the orientation of this axis reduces the potential six-dimensional search to four (Eulerian angle gamma and three translational parameters). The direct four-dimensional search within the unit cell produced a clear solution. The electron-density map based on this solution agrees well with the known structure, and the phase error calculated from the map was 61 degrees within 20 A resolution. It is anticipated that the low-resolution envelope can be used as a starting model for phase extension by the maximum-entropy and density-modification method.

Crystallography, X-Ray↗
PubMed1999

Biochemical characterization and solution structure of nitrous oxide reductase from Alcaligenes xylosoxidans (NCIMB 11015).

Nitrous oxide reductase (N2OR) is the terminal enzyme involved in denitrification by microbes. No three-dimensional structural information has been published for this enzyme. We have isolated and characterised N2OR from Alcaligenes xylosoxidans (AxN2OR) as a homodimer of M(r) 134,000 containing seven to eight copper atoms per dimer. Comparison of sequence and compositional data with other N2ORs suggests that AxN2OR is typical and can be expected to have similar domain folding and subunit structure to other members of this family of enzymes. We present synchrotron X-ray-scattering data, analysed using a model-independent method for shape restoration, which gave a approximately 20 A resolution structure of the enzyme in solution, providing a glimpse of the structure of any N2OR and shedding light on the molecular architecture of the molecule. The specific activity of AxN2OR was approximately 6 mumol of N2O reduced.min-1. (mg of protein)-1; N2OR activity showed both base and temperature activation. The visible spectrum exhibited an absorption maximum at 550 nm with a shoulder at 635 nm. On oxidation with K3Fe(CN)6, the absorption maximum shifted to 540 nm and a new shoulder at 480 nm appeared. Reduction under anaerobic conditions resulted in the formation of an inactive blue form of the enzyme with a broad absorption maximum at 650 nm. As isolated, the enzyme shows an almost featureless EPR spectrum, which changes on oxidation to give an almost completely resolved seven-line hyperfine signal in the gII region, g = 2.18, with AII = 40 G, consistent with the enzyme being partially reduced as isolated. Both the optical and EPR spectra of the oxidized enzyme are characteristic of the presence of a CuA centre.

Alcaligenes↗
PubMed1999

Is single-wavelength anomalous scattering sufficient for solving phases? A comparison of different methods for a 2.1 A structure solution.

The structure of rusticyanin is the largest unknown structure (M(r) = 16.8 kDa) which has been recently solved by the direct-methods approach using only single-wavelength anomalous scattering (SAS) data from the native protein [Harvey et al. (1998). Acta Cryst. D54, 629-635]. Here, the results of the Sim distribution approach [Hendrickson & Teeter (1981). Nature (London), 290, 107-113] and of the CCP4 procedure MLPHARE [Collaborative Computational Project, Number 4 (1994). Acta Cryst. D50, 760-763] are compared with those from direct methods. Analysis against the final refined model shows that direct methods produced significantly better phases (average phase error 56 degrees ) and therefore significantly better electron-density maps than the Sim distribution and MLPHARE approaches (average phase error was around 63 degrees in both cases).

Azurin↗
PubMed1998

X-ray structure of a blue-copper nitrite reductase in two crystal forms. The nature of the copper sites, mode of substrate binding and recognition by redox partner.

Denitrification is one of the main steps of the global nitrogen cycle that is sustained by prokaryotic organisms. Denitrifying bacteria use two entirely different enzymes in this process, one based on haem cd1 prosthetic groups and the other on type 1-type 2 Cu centres. Copper-containing nitrite reductases (NiRs) are sub-divided into blue and green NiRs, which are respectively thought to be redox partners of azurins and pseudo-azurins. Crystallographic structures of the blue nitrite reductase from Alcaligenes xylosoxidans (AxNiR) are presented in the oxidised hexagonal form and the substrate-bound orthorhombic form to 2.1 A and 2.8 A resolution, respectively. The complete amino acid sequence of AxNiR has been determined by conventional chemical analysis. A 3 A structure of AxNiR has been published where the modelling was based on the sequence of another blue NiR. The higher resolution of the hexagonal form together with the correct sequence allows a detailed comparison with the crystallographic structures of the green NiRs. There is a striking difference in the overall surface charge distribution between the two sub-groups, providing a neat structural explanation for their different reactivities to pseudoazurin or azurin and supporting the view that electron transfer proceeds via complex formation. A detailed examination of the type-1 Cu site, the site responsible for the colour, reveals several subtle differences, including a lateral displacement of 0.7 A for Smet. The structure of the type-2 Cu site, and changes that occur upon substrate binding are discussed in terms of the catalytic mechanism. The similarity of the type 2 Cu site to the catalytic Zn site in carbonic anhydrase and the catalytic Cu site of superoxide dismutase is re-examined in view of the high-resolution (2.1 A) structure.

Alcaligenes↗
PubMed1998

Modulating the redox potential and acid stability of rusticyanin by site-directed mutagenesis of Ser86.

The expression of rusticyanin in Escherichia coli and a number of mutants for Ser86 is reported. Mutations of Ser86 to Asn, Asp, Gln, and Leu were undertaken as this is an Asn residue in other structurally characterized cupredoxins, and it has been suggested that this may be partly responsible for the high redox potential (680 mV) and extreme acid stability of rusticyanin. N-Terminal sequence analysis, together with other biochemical and spectrochemical characterization, shows that the recombinant wild-type protein is indistinguishable from native rusticyanin. All four mutants retain the rhombic nature of the EPR spectra and a significant absorption maximum at approximately 450 nm, thus confirming that the overall geometry of the Cu ligands is essentially maintained. The oxidized form of all four mutants is less acid stable than the wild-type protein, although the detailed mechanism of lability varies. Ser86Leu readily loses copper as the pH is reduced from 4.0, but the protein does not denature. A significant proportion (approximately 30%) of Ser86Gln is denatured at lower pH values, whereas Ser86Asn and Ser86Asp are stable as the reduced (CuI) protein. The redox potential also varies by approximately 110 mV (590-702 mV) upon these single point mutations, thus providing direct experimental support to the idea that this residue is at least in part responsible for the acid stability and the highest redox potential of rusticyanin in the cupredoxin family.

Amino Acid Sequence↗
PubMed1998

Structure determination of a 16.8 kDa copper protein at 2.1 A resolution using anomalous scattering data with direct methods.

The structure of rusticyanin, an acid-stable copper protein, has been determined at 2.1 A resolution by direct methods combined with the single-wavelength anomalous scattering (SAS) of copper (f" = 3.9 e-) and then conventionally refined (Rcryst = 18.7%, Rfree = 21.9%). This is the largest unknown protein structure (Mr approximately /= 16.8 kDa) to be determined using the SAS and direct-methods approach and demonstrates that by exploiting the anomalous signal at a single wavelength, direct methods can be used to determine phases at typical (approximately 2 A) macromolecular crystallographic resolutions. Extrapolating from the size of the anomalous signal for copper (f" approximately 4 e-), this result suggests that the approach could be used for proteins with molecular weights of up to 33 kDa per Se (f"max++ = 8 e- at the 'white line') and 80 kDa for a Pt derivative (f"max = 19 e- at the 'white line', L3 edge). The method provides a powerful alternative in solving a de novo protein structure without either preparing multiple crystals (i.e. isomorphous heavy-atom derivative plus native crystals) or collecting multi-wavelength anomalous diffraction (MAD) data.

Azurin↗
PubMed1998

The nature of ligand-induced conformational change in transferrin in solution. An investigation using X-ray scattering, XAFS and site-directed mutants.

Ligand-induced conformational change in transferrins has been studied by site-directed mutagenesis of human serum half molecule (N-lobe), X-ray absorption fine structure (XAFS) spectroscopy and X-ray solution scattering. Use of recent advances in data analysis has been made for extracting model-independent molecular shapes from X-ray solution scattering data for the intact, the half molecule and its mutants. Clear evidence is provided that the transferrin molecule (intact as well as N-lobe), in its apo and holo forms, exists for the majority of the time in well-defined specific conformations representing the "fully opened" and "closed" states of the molecule, respectively. Evidence is also provided for the existence of an additional conformation, referred to here as the "intermediate" conformation for simplicity, which is trapped in the case of some of the mutants in the iron-bound form. We suggest that domain closure in the transferrin molecule is a two-step process, with the intermediate conformation representing the first stage of domain closure (approximately 20 degrees hinge-twist of domain II). Our data are not inconsistent with the ligand-free molecule sampling the closed states occasionally (< or = 10%) but are not in support of a continuous conformational search between the fully opened and closed states in the absence of iron.

Animals↗
PubMed1997

Structures of a blue-copper nitrite reductase and its substrate-bound complex.

Copper-containing nitrite reductases (NiR's) have been conveniently subdivided into blue and green NiR's which are thought to be redox partners of azurins and pseudo-azurins, respectively. Crystal structures of two green NiR's have recently been determined. Alcaligenes xylosoxidans has been shown to have a blue-copper nitrite reductase (AxNiR) and two azurins with 67% homology both of which donate electrons to it effectively. The first crystal structure of a blue NiR (AxNiR) in its oxidized and nitrite-bound forms, with particular emphasis to the Cu sites, is presented. The Cu-Smet distance is the same as those in the green NiR's. Thus, the length of this interaction is unlikely to be responsible for differences in colour. Crystallographic data presented here taken together with structural data of other single Cu type-1 proteins and their mutants suggest that the displacement of Cu from the strong ligand plane is perhaps the cause for the differences in colour observed for otherwise 'classical' blue Cu centre. Nitrite is observed binding to the catalytic Cu in a bidentate fashion displacing the water molecule, offering a neat rationalization for the XAFS observation that the type-2 Cu-ligand distances increase on nitrite binding as a result of increased coordination. These results are discussed in terms of enzyme mechanism.

Journal Article↗
PubMed1997

A critical assessment of the evidence from XAFS and crystallography for the breakage of the imidazolate bridge during catalysis in CuZn superoxide dismutase.

BACKGROUND: Copper-zinc superoxide dismutase (CuZn SOD) protects cells from the toxic effects of superoxide radicals. Key steps in the catalytic mechanism of CuZn SOD are thought to be the breakage of the imidazolate bridge between copper and zinc upon reduction of the copper site and the subsequent proton donation from the bridging histidine. This view has been recently challenged by a crystallographic study at 1.9 A resolution where evidence for a five-coordinate copper site in the reduced enzyme was provided. In contrast, a crystallographic study of yeast CuZn SOD at 1.7 A has confirmed the breaking of the bridging histidine in reduced crystals. We have examined the nature of the changes in metal coordination which result upon reduction of the enzyme using the X-ray absorption fine structure (XAFS) technique. RESULTS: The copper and zinc K-edge XAFS data of bovine SOD, recorded in the buffer systems used in the two crystallographic studies, were analyzed by constrained refinement using fast curved wave theory, taking full account of multiple-scattering effects. The study confirms that in the oxidized form of the enzyme the copper atom is five coordinate, with four histidine ligands at 1.99 +/- 0.02 A and a water molecule at 2.18 +/- 0.03 A. In the reduced form of the enzyme, one of the histidine ligands and the water molecule are lost from the inner coordination sphere of the copper, with the three remaining histidines ligated at 1.97 +/- 0.02 A. The X-ray absorption near edge structure (XANES) of the reduced enzyme is consistent with an approximate trigonal planar geometry at the copper site. The XAFS at the zinc K-edge is essentially identical in both the oxidized and reduced enzyme and is accounted for by three histidines coordinated at 2.01 +/- 0.02 A and an aspartate ligand at 1.96 +/- 0.03 A. CONCLUSIONS: The existence of a three-coordinate cuprous ion in bovine CuZn SOD is demonstrated and is a key feature of catalytic degradation of superoxide substrate by SOD involving alternate Cu(I) and Cu(II) states of the enzyme. Only subtle changes in the zinc K-edge XAFS take place upon reduction. Thus the reaction mechanism which involves breakage of the bridging histidine is unambiguously supported by the XAFS data.

Animals↗
PubMed1997

The first glimpse of a complex of nitrogenase component proteins by solution X-ray scattering: conformation of the electron transfer transition state complex of Klebsiella pneumoniae nitrogenase.

An essential feature of the mechanism of nitrogenase, the enzyme responsible for biological nitrogen fixation, is the formation of a transient electron transfer complex between the MoFe protein containing the active site at which N2 is reduced, and the Fe protein, which functions as a specific electron donor to the MoFe protein. We have obtained high quality solution X-ray scattering data using synchrotron X-rays of a stable putative electron transfer complex, (MoFe-protein)(Fe-protein.ADP.AIF4)2, of Klebsiella pneumoniae and used the model-independent approach based on the multipole expansion method to provide a stable and unique shape restoration at approximately 15 A resolution. The biological significance of this first molecular structure of a nitrogenase complex is discussed.

Azotobacter vinelandii↗
PubMed1996

Effect of pH and ligand binding on the structure of the Cu site of the Met121Glu mutant of azurin from Pseudomonas aeruginosa.

A pH-dependent X-ray absorption fine structure (XAFS) study has been undertaken to provide a structural interpretation of the spectroscopic properties of the Met121 Glu mutant of azurin from Pseudomonas aeruginosa (Azp). Ligand binding studies have been carried out to investigate the effect of the cavity formed at the Cu site as a result of the mutation. The optical spectrum at pH 4 exhibits an intense band at approximately 600 nm and a weaker band at approximately 450 nm, typical for the blue copper proteins. As the pH is increased, these bands decrease in intensity and shift to 570 and 413 nm, respectively, with the latter becoming the more intense of the two [Karlsson, B.G., et al. (1991) Protein Eng. 4 (3), 343-349]. These changes are accompanied by a change in the EPR spectrum from a rhombic type 1 Cu spectrum at pH 4 to a spectrum with the rhombic splitting decreasing to zero and the hyperfine coupling increasing from 25 to 83 G. X-ray absorption a the Cu K-edge shows that this change results from the lengthening of the Cu-His (by 0.07 A) and Cu-Cys (by 0.06 A) bonds and the coordination of one of the oxygen atoms of the glutamate ligand at pH 8, at a distance as close as 1.90 A. The copper site thus changes from a normal type 1 copper center with three strong bonds at pH 4 to a copper site with four strong bonds at pH 8, with Cu-His distances significantly longer than known distances for type 1 copper centres measured using the XAFS technique. The XAFS of the azide derivative measured at pH 8 shows a similar Cu coordination, with azide replacing glutamate as the fourth ligand. Azide binding at pH 8 is accompanied by a further increase in the EPR hyperfine coupling to 110 G. This structural information when taken together with recent structural sudies on copper proteins points toward the need for a reexamination of the basis on which copper proteins are classified.

Absorptiometry, Photon↗
PubMed1996

Anaerobic crystallisation of an isopenicillin N synthase.Fe(II).substrate complex demonstrated by X-ray studies.

Isopenicillin N synthase (IPNS) was cocrystallised with ferrous sulphate and its substrate, delta-(L-alpha-aminoadipoyl)-L-cysteinyl-D-valine (Aad-Cys-Val). Vital to the successful procedure was the maintenance of a rigorously anaerobic environment. Hanging-drop vapour-diffusion crystallisation experiments, using lithium sulphate as the precipitant produced three crystal forms. Form I crystals, with a plate habit, diffracted X-rays to at least 0.11-nm resolution at the European Synchrotron Radiation Facility and belong to the space group P2(1)2(1)2(1), with unit-cell dimensions a = 4.68, b = 7.15, c = 10.10 nm. Their asymmetric unit contains a single IPNS.Fe(II).Aad-Cys-Val complex with a solvent content of 38.5%. Form II crystals, with a hexagonal habit, diffract X-rays to at least 0.21 nm resolution at the European Synchrotron Radiation Facility and belong to the space group P3(1)21, with unit-cell dimensions a = 10.10, b = 10.10, c = 11.567 nm. Their asymmetric unit also contains a single IPNS.Fe(II).Aad-Cys-Val complex with a solvent content of 69.5%. Form III crystals, needles, do not show well-ordered diffraction. Although all three forms were initially produced in crystallisation experiments under identical conditions, appropriate micro and streak seeding allows selective crystallisation of form I or form II crystals. Extended X-ray-absorption fine-structure studies on a crystalline slurry of the form I crystals demonstrate the presence of an Fe-S(Aad-Cys-Val) bond length of 0.234 +/- 0.003 nm.

Anaerobiosis↗