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R R Eady

Publications and source records attributed to R R Eady.

At least 37 records · Page 2Linked to original sources
PubMed1998

The intramolecular electron transfer between copper sites of nitrite reductase: a comparison with ascorbate oxidase.

The intramolecular electron transfer (ET) between the type 1 Cu(I) and the type 2 Cu(II) sites of Alcaligenes xylosoxidans dissimilatory nitrite reductase (AxNiR) has been studied in order to compare it with the analogous process taking place in ascorbate oxidase (AO). This internal process is induced following reduction of the type 1 Cu(II) by radicals produced by pulse radiolysis. The reversible ET reaction proceeds with a rate constant kET = k(1-->2) + k(2-->1) of 450 +/- 30 s(-1) at pH 7.0 and 298 K. The equilibrium constant K was determined to be 0.7 at 298 K from which the individual rate constants for the forward and backward reactions were calculated to be: k(1-->2) = 185 +/- 12 s(-1) and k(2-->1) 265 +/- 18 s(-1). The temperature dependence of K allowed us to determine the deltaH(o) value of the ET equilibrium to be 12.1 kJ mol(-1). Measurements of the temperature dependence of the ET process yielded the following activation parameters: forward reaction, deltaH* = 22.7 +/- 3.4 kJ mol(-1) and deltaS* = -126 +/- 11 J K(-1) mol(-1); backward reaction, deltaH* = 10.6 +/- 1.7 kJ mol(-1) and deltaS* = -164 +/- 15 J K(-1) mol(-1). X-ray crystallographic studies of NiRs suggest that the most probable ET pathway linking the two copper sites consists of Cys136, which provides the thiolate ligand to the type 1 copper ion, and the adjacent His135 residue with its imidazole being one of the ligands to the type 2 Cu ion. This pathway is essentially identical to that operating between the type 1 Cu(I) and the trinuclear copper centre in ascorbate oxidase, and the characteristics of the internal ET processes of these enzymes are compared. The data are consistent with the faster ET observed in nitrite reductase arising from a more advantageous entropy of activation when compared with ascorbate oxidase.

Alcaligenes↗
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

Nucleotide binding by the nitrogenase Fe protein: a 31P NMR study of ADP and ATP interactions with the Fe protein of Klebsiella pneumoniae.

Investigation of the interaction of MgADP- and MgATP2- with the Fe protein of Klebsiella pneumoniae nitrogenase by 31P NMR showed that the adenine nucleotides are reversibly bound in slow exchange with free nucleotides. Dissociation of the MgADP--Fe protein complex was slow enough to enable its isolation by gel filtration, thus permitting the assignment of resonances to bound nucleotides. Spectra of ADP bound to Kp2 were similar to spectra of ADP bound to the myosin motor domain. Oxidative inactivation of a Kp2-MgADP- complex with excess ferricyanide ion eliminated exchange between bound and free ADP, indicating that the intact iron sulphur cluster, located 20 A from the binding sites, is required for the reversible binding of MgADP-. A change in conformation on controlled oxidation of Kp2 with indigocarmine increased the chemical shift of the beta phosphate resonance of bound MgADP-. Both oxidized and reduced conformers were observed transiently in the absence of dithionite. The 31P resonances of both the beta and gamma phosphates of bound MgATP2- indicated major changes in environment and labilization of both groups on binding to the Fe protein. Highly purified Kp2 slowly hydrolysed ATP, resulting in mixtures of bound nucleotides. Partial occupation of Kp2 MgATP2--binding sites (N=1.9+/-0.2, Kd=145 microM) in concentrated protein solutions was demonstrated by flow dialysis. Scatchard plots of data for bound and free ligand obtained after equilibration with Kp2 were linear and no co-operative interactions were detected. We conclude that MgADP- stabilizes the oxidized Fe protein conformer and this conformation in turn triggers the dissociation of the Fe protein from the MoFe protein in the rate-limiting step of the overall process of dinitrogen reduction.

Adenosine Diphosphate↗
PubMed1997

Nitrogenase of Klebsiella pneumoniae: kinetics of formation of the transition-state complex and evidence for an altered conformation of MoFe protein lacking a FeMoco centre.

We have investigated the kinetics of inactivation of Mo-nitrogenase isolated from Klebsiella pneumoniae when it forms an inhibited putative transition-state complex on incubation with ADP and AlF4-. In the presence of excess Kp2 (Fe protein of the Mo-nitrogenase of K. pneumoniae), the kinetics were found to depend on the Mo content of Kp1 (the MoFe protein of Mo-nitrogenase of K. pneumoniae). The residual nitrogenase activity versus time of incubation using Kp1 preparations containing integral, i.e. one or two Mo atoms per molecule of Kp1, were essentially monophasic, but significantly different rates of inactivation were observed. In contrast, the progress curves for preparations of Kp1 with non-integral Mo content were biphasic, suggesting the presence of two discrete catalytically active species of Kp1. The best fit to the observed data was obtained with a two-exponential expression, the amplitude of which was consistent with the Mo content, provided that the fast phase of the reaction was assigned to a Kp1 species containing one, and the slow phase to a species containing two Mo atoms per alpha2beta2 tetramer. This analysis provides the first evidence for the existence of a catalytically active Kp1 species containing a single Mo atom. These data also indicate that MoFe protein which does not have all FeMoco binding sites occupied has an altered conformation compared with a fully loaded protein, and that the Fe protein reacts with these conformations at different rates to form the stable, but inhibited transition-state complex.

Enzyme Activation↗
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

pH-dependence for binding a single nitrite ion to each type-2 copper centre in the copper-containing nitrite reductase of Alcaligenes xylosoxidans.

The first quantitative characterization of the interaction of NO2(-) with the Cu-containing dissimilatory nitrite reductase (NiR) of Alcaligenes xylosoxidans using steady-state kinetics, equilibrium gel filtration and EPR spectroscopy is described. Each molecule of this protein consists of three equivalent subunits, each containing a type-1 Cu atom and also a type-2 Cu atom at each subunit interface. Enzyme activity increased in a biphasic manner with decreasing pH, having an optimum at pH 5.2 and a plateau between pH 6.1 and 5.8. Equilibrium gel filtration showed that binding of NO2(-) to the oxidized NiR was also pH-dependent. At pH 7.5, no binding was detectable, but binding was detectable at lower pH values. At pH 5.2, the concentration-dependence for binding of NO2(-) to the enzyme showed that approx. 4.1 NO2(-) ions bound per trimeric NiR molecule. Unexpectedly, NiR deficient in type-2 Cu centres bound 1.3 NO2(-) ions per trimer. When corrected for this binding, a value of 3 NO2(-) ions bound per trimer of NiR, equivalent to the type-2 Cu content. The NO2(-)-induced changes in the EPR parameters of the type-2 Cu centre of the oxidized enzyme showed a similar pH-dependence to that of the activity. Binding constants for NO2(-) at a single type of site, after allowing for the non-specifically bound NO2(-), were 350+/-35 microM (mean+/-S.E.M.) at pH 7.5 and <30 microM at pH 5.2. The apparent Km for NO2(-) with saturating concentrations of dithionite as reductant was 35 microM at pH 7.5, which is 10-fold tighter than for the oxidized enzyme, and is compatible with an ordered mechanism in which the enzyme is reduced before NO2(-) binds.

Alcaligenes↗
PubMed1997

Covalent modification of nitrogenase MoFe protein by ADP.

MgADP- reacted with the nitrogenase molybdenum-iron (MoFe) protein of Klebsiella pneumoniae (Kp1) over a period of 2 h to yield a stable, catalytically active conjugate. The isolated protein exhibited a new, broad 31P NMR resonance at -1 p.p.m. lacking phosphorus J coupling. The adenine ring of [8-14C]ADP remained associated with the conjugate. A covalently bound nucleotide was identified as AMP by NMR and TLC. Extended dialysis of Kp1 against MgADP- resulted in further AMP binding at the protein surface. ADP was initially bound tightly to Kp1 at a site distinct from the AMP sites. ATP did not replace ADP. The time course of the formation of the Kp1-AMP was altered by the nitrogenase iron protein (Kp2) and was dependent on redox potential. Kp1-AMP was stable to concentration and oxidation with ferricyanide ion at -350 mV. Slow hydrolysis of Kp1-AMP over a period of 6 h yielded AMP and unaltered Kp1. The adenine ring of ADP exchanged with adenine of MgATP2- during reductant-limited turnover of nitrogenase under N2, indicating reversibility of ATP hydrolysis at 15 degrees C. [32P]Pi exchanged with the terminal phosphate group of both ADP and ATP on incubation with Kp1. 32P exchange and the catalytic activity of Kp1 were inhibited by a 20-fold molar excess of the lysine-modifying reagent, o-phthalaldehyde (OPT). Preincubation with MgADP- protected against OPT inactivation. Two potentially reactive lysine residues on the alpha chain of the MoFe protein near a putative hydrophobic docking site for the nitrogenase Fe protein are proposed as sites of OPT and nucleotide binding. Azotobacter vinelandii MoFe protein (Av1) also formed an AMP adduct but Kp2 did not. Catalase did not interact with ADP. The reactions of the nitrogenase MoFe protein with adenine nucleotides have no counterpart in known protein-nucleotide interactions.

Adenosine Diphosphate↗
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

Flavodoxin 1 of Azotobacter vinelandii: characterization and role in electron donation to purified assimilatory nitrate reductase.

Flavodoxins synthesized by Azotobacter vinelandii strain UW 36 during growth on nitrate as nitrogen source were separated by FPLC on a Mono Q column into two species, flavodoxin 1 (AvFld 1) and flavodoxin 2 (AvFld 2). Both proteins migrated as single bands on SDS/PAGE. AvFld 1 was approx. 5-fold more abundant than AvFld 2 in the unresolved flavodoxin mixture. N-terminal amino acid analysis showed the sequence of AvFld 2 to correspond to the nif F gene product, an electron donor to nitrogenase. The sequences also show that these species corresponded to the flavodoxins Fld A and Fld B isolated from N2-grown cultures of the closely related organism Azotobacter throococcum [Bagby, Barker, Hill, Eady and Thorneley (1991) Biochem.J.277, 313-319]. Electrospray mass spectrometry gave M, values for the polypeptides of 19430 +/- 3 and 19533 +/- 5 respectively. 31P-NMR measurements showed that in addition to the phosphate associated with the FMN (delta = -136.3 p.p.m. and -135.48 p.p.m.), AvFld 1 had a signal at delta = -142.1 p.p.m. and AvFld 2 at delta = -138.59 p.p.m. present in substoichiometric amounts with FMN. These appeared to arise from unstable species since they were readily lost on further manipulation of the proteins. The mid-point potentials of the semiquinone hydroquinone redox couples were -330 mV and -493 mV for AvFld 1 and AvFld 2 respectively, but only AvFld 1 was competent in donating electrons to the purified assimilatory nitrate reductase of A. vinelandii to catalyse the reduction of nitrate to nitrite. Flavodoxin isolated from NH4(+)-grown cells (Fld 3) also functioned as electron donor at half the rate of AvFld 1, but ferredoxin 1 from A. chroococcum did not.

Amino Acid Sequence↗
PubMed1996

Structure Solution of Azurin II from Alcaligenes xylosoxidans using the Laue Method: Possibility of Studying In Situ Redox Changes using X-rays.

We have recently demonstrated that X-rays can be used for changing the redox states of the metal centre in metalloproteins [Murphy et al. (1995). J. Synchrotron Rad. 2, 64-69]. The possibility of using the Laue method for studying the structural changes associated with such X-ray-induced reactions is explored by applying the method to the structure determination of a new azurin (hereafter referred to as azurin II) from the denitrifying bacterium Alcaligenes xylosoxidans. Laue X-ray diffraction data of azurin II were collected at station 9.7 of the SRS Daresbury. Three diffraction patterns were recorded on film packs at three different crystal orientations. The data were processed using the Daresbury Laue Software Suite to give 2224 independent single reflections (R(merge) = 0.136) in the wavelength range 0.36-1.40 A. The data completeness was 44% at 2.55 A resolution. Phase determination for the data was undertaken using the molecular-replacement method; the top peak was chosen in both the rotation function and the subsequent translation function. This solution agreed well with the molecular-replacement solution achieved independently using monochromatic data. The electron-density map showed reasonably good agreement with the model and the copper site was readily recognizable as it had the highest density. To see if the electron-density map could be improved, ;the doublets in the diffraction data were then deconvoluted. This added 26% data in the region infinity-2d(min) resulting in an improvement in the data completeness to 50% and thus in improved continuity of the electron-density map. The quality of these maps is discussed from the point of view of the suitability of this approach for studying redox-induced structural changes.

Journal Article↗
PubMed1996

Possible role of a short extra loop of the long-chain flavodoxin from Azotobacter chroococcum in electron transfer to nitrogenase: complete 1H, 15N and 13C backbone assignments and secondary solution structure of the flavodoxin.

The 1H, 15N and 13C backbone and 1H and 13C beta resonance assignments of the long-chain flavodoxin from Azotobacter chroococcum (the 20-kDa nifF product, flavodoxin-2) in its oxidized form were made at pH 6.5 and 30 degrees C using heteronuclear multidimensional NMR spectroscopy. Analysis of the NOE connectivities, together with amide exchange rates, 3JHNH alpha coupling constants and secondary chemical shifts, provided extensive solution secondary structure information. The secondary structure consists of a five-stranded parallel beta-sheet and five alpha-helices. One of the outer regions of the beta-sheet shows no regular extended conformation, whereas the outer strand beta 4/6 is interrupted by a loop, which is typically observed in long-chain flavodoxins. Two of the five alpha-helices are nonregular at the N-terminus of the helix. Loop regions close to the FMN are identified. Negatively charged amino acid residues are found to be mainly clustered around the FMN, whereas a cluster of positively charged residues is located in one of the alpha-helices. Titration of the flavodoxin with the Fe protein of the A. chroococcum nitrogenase enzyme complex revealed that residues Asn11, Ser68 and Asn72 are involved in complex formation between the flavodoxin and Fe protein. The interaction between the flavodoxin and the Fe protein is influenced by MgADP and is of electrostatic nature.

Amino Acid Sequence↗
PubMed1995

Structure of a new azurin from the denitrifying bacterium Alcaligenes xylosoxidans at high resolution.

It has been reported previously that Alcaligenes xylosoxidans (NC1MB 11015) grown under denitrifying conditions produces two azurins instead of the single previously identified azurin [Dodd, Hasnain, Hunter, Abraham, Debenham, Kanzler, Eldridge, Eady, Ambler & Smith (1995). Biochemistry. In the press]. The new azurin, called azurin II, has been crystallized as blue elongated rectangular prisms with the tetragonal space group P4(1)22 and unit-cell parameters a = b = 52.65, c = 100.63 A. X-ray crystallographic data extending to 1.9 A resolution were collected by the Weissenberg method using 200 x 400 mm image plates and synchrotron X-rays of wavelength 0.97 A. The three-dimensional structure of azurin II has been solved by the molecular-replacement method using the structure of azurin from Alcaligenes denitrificans NCTC 8582 with which this new azurin shows a close homology. The quality of the initial map was sufficient to predict a number of sequence differences. The model is currently refined to an R-factor of 18.8% with X-ray data between 8.5 and 1.9 A. The final model of 961 protein atoms, one Cu atom and 50 water molecules has r.m.s. deviations from ideality of 0.009 A for bond lengths and 1.7 degrees for bond angles. The overall structure is similar to that of the azurin from A. denitrificans NCTC 8582. It has a beta-barrel structure with the Cu atom located near the top end of the molecule. The Cu atom is coordinated to Ndelta of His46 and His117 at 2.02 A and to Sgamma of Cys112 at 2.12 A, while the carbonyl O atom of Gly45 and Sdelta atom of Met121 provide the additional interactions at 2.75 and 3.26 A, respectively.

Journal Article↗
PubMed1995

Evidence for two distinct azurins in Alcaligenes xylosoxidans (NCIMB 11015): potential electron donors to nitrite reductase.

We have isolated two type 1 copper-containing proteins (M(r) approximately 13K) from Alcaligenes xylosoxidans (NCIMB 11015) grown under denitrifying conditions. Amino acid sequence analysis of these two proteins shows one to be the previously identified azurin (Ambler, 1971), which we shall call azurin I, and the other to be a related, but previously undescribed, blue copper protein which we show to also be an azurin and propose to call azurin II. Thus, NCIMB 11015 becomes the second system where two distinct azurins are found, the other being Methylomonas J (Ambler & Tobari, 1989). On isoelectric focusing, azurin I migrates very similarly to the previously identified azurin from this organism while azurin II migrates similarly to azurin purified from Alcaligenes denitrificans NCTC 8582. The sequence of azurin II is 33% different than the azurin I sequence but is only 11% different than the azurin from Alcaligenes denitrificans NCTC 8582. Optical spectra for the two proteins are very similar with epsilon mM values of 6.27 and 5.73 mM-1 cm-1 for azurin I and II, respectively, at lambda max approximately 620 nm. The 291 nm shoulder normally ascribed to the hydrophobic nature of tryptophan 48 is clearly observed in azurin I but is missing in azurin II. Amino acid analysis confirms that this tryptophan is missing in azurin II. Azurin I and azurin II show essentially the same redox potential of 305 +/- 10 mV at pH 7.5 and are equally effective electron donors to the purified dissimilatory nitrite reductase of Alc. xylosoxidans in vitro.(ABSTRACT TRUNCATED AT 250 WORDS)

Alcaligenes↗
PubMed1995

The substrate-binding site in Cu nitrite reductase and its similarity to Zn carbonic anhydrase.

Here we investigate the structure of the two types of copper site in nitrite reductase from Alcaligenes xylosoxidans, the molecular organisation of the enzyme when the type-2 copper is absent, and its mode of substrate binding. X-ray absorption studies provide evidence for a fourth ligand at the type-2 Cu, that substrate binds to this site and indicates that this binding does not change the type-1 Cu centre. The substrate replaces a putative water ligand and is accommodated by a lengthening of the Cu-histidine bond by approximately 0.08 A. Modelling suggests a similarity between this unusual type-2 Cu site and the Zn site in carbonic anhydrase and that nitrite is anchored by hydrogen bonds to an unligated histidine present in the type-2 Cu cavity.

Absorptiometry, Photon↗
PubMed1994

EPR and electron nuclear double resonance (ENDOR) studies show nitrite binding to the type 2 copper centers of the dissimilatory nitrite reductase of Alcaligenes xylosoxidans (NCIMB 11015).

EPR and 1H, 14,15N ENDOR spectra are described for the type 1 and type 2 Cu(II) centers of dissimilatory nitrite reductase (NiR) from Alcaligenes xylosoxidans. The study was carried out on preparations of NiR containing both type 1 and type 2 Cu sites, and also on preparations of lower activity which contained essentially only type 1 Cu centers. This has enabled ENDOR studies of type 1 and type 2 sites to be carried out largely independently of each other, by appropriate choice of the excitation field. Spectra were recorded both in the absence and presence of nitrite, allowing a clear determination of which of the two types of Cu center constitutes the substrate binding site. The EPR results show large changes in the type 2 site gparallel (which decreases by 0.065) and CuAparallel (which increases by 2.0 mT) while the type 1 site EPR is not affected. In addition, both 1H and 14N ENDOR of the type 2 Cu site undergo considerable changes on addition of nitrite whereas the type 1 Cu site ENDOR is unaffected. Our results clearly demonstrate that nitrite binds to the type 2 copper and that this process significantly perturbs the ligation of this copper by the protein histidine residues. No 15N ENDOR resonances were observed from 15N nitrite. The accessibility of the copper sites to solvent has been studied using 2H2O. The results indicate that nitrite binds to the type 2 Cu by displacing a proton, probably on a water molecule bound to the copper atom.

Alcaligenes↗
PubMed1993

X-ray scattering using synchrotron radiation shows nitrite reductase from Achromobacter xylosoxidans to be a trimer in solution.

We demonstrate here the applicability of X-ray scattering for studying molecular conformation of multimeric proteins in solution by using synchrotron radiation to extend the range of data collection to include medium angles (ca. 3-4 degrees). We have been able to define the solution structure of the dissimilatory nitrite reductase of Achromobacter xylosoxidans (AxNiR), an enzyme for which there are conflicting reports as to the nature of its multimeric structure. Quantitative interpretation of the X-ray scattering profile, based on a modeling study using the high-resolution crystal structure data for the nitrite reductase from the related organism Achromobacter cycloclastes (AcNiR), provides a detailed model for the trimeric structure of AxNiR in solution. Sedimentation equilibrium centrifugation gave an M(r) of 103,000, consistent with such a trimeric structure.

Alcaligenes↗