Biochemical and crystallographic studies of the Met144Ala, Asp92Asn and His254Phe mutants of the nitrite reductase from Alcaligenes xylosoxidans provide insight into the enzyme mechanism (original) (raw)
Related papers
Biochemistry, 2002
Nitrite reductase of Alcaligenes xylosoxidans contains three blue type 1 copper centers with a function in electron transfer and three catalytic type 2 copper centers. The mutation H139A, in which the solvent-exposed histidine ligand of the type 1 copper ion was changed to alanine, resulted in the formation of a colorless protein containing 4.4 Cu atoms per trimer. The enzyme was inactive with reduced azurin as the electron donor, and in contrast to the wild-type enzyme, no EPR features assignable to type 1 copper centers were observed. Instead, the EPR spectrum of the H139A enzyme, with parameters of g 1) 2.347 and A 1) 10 mT, was typical of type 2 copper centers. On the addition of nitrite, the EPR features developed spectral features with increased rhombicity, with g 1) 2.29 and A 1) 11 mT, arising from the type 2 catalytic site. As assessed by visible spectroscopy, ferricyanide (E°) +430 mV) was unable to oxidize the H139A enzyme, and this required a 30-fold excess of K 2 IrCl 6 (E°) +867 mV). Oxidation resulted in the EPR spectrum developing additional axial features with g 1) 2.20 and A 1) 9.5 mT, typical of type 1 copper centers. The oxidized enzyme after separation from the excess of K 2 IrCl 6 by gel filtration was a blue-green color with absorbance maxima at 618 and 420 nm. The instability of the protein prevented the precise determination of the midpoint potential, but these properties indicate that it is in the range 700-800 mV, an increase of at least ∼470 mV compared with the native enzyme. This high potential, which is consistent with a trigonal planar geometry of the Cu ion, effectively prevents azurin-mediated electron transfer from the type 1 center to the catalytic type 2 Cu site. However, with dithionite as reductant, 20% of the activity of the wild-type enzyme was observed, indicating that the direct reduction of the catalytic site by dithionite can occur. When CuSO 4 was added to the crude extract before isolation of the enzyme, the Cu content of the purified H139A enzyme increased to 5.7 Cu atoms per trimer. The enzyme remained colorless, and the activity with dithionite as a donor was not significantly increased. The additional copper in such preparations was associated with an axial type 2 Cu EPR signal with g 1) 2.226 and A 1) 18 mT, and which were not changed by the addition of nitrite, consistent with the activity data.
Biochemistry, 2002
Nitrite reductase of Alcaligenes xylosoxidans contains three blue type 1 copper centers with a function in electron transfer and three catalytic type 2 copper centers. The mutation H139A, in which the solvent-exposed histidine ligand of the type 1 copper ion was changed to alanine, resulted in the formation of a colorless protein containing 4.4 Cu atoms per trimer. The enzyme was inactive with reduced azurin as the electron donor, and in contrast to the wild-type enzyme, no EPR features assignable to type 1 copper centers were observed. Instead, the EPR spectrum of the H139A enzyme, with parameters of g 1) 2.347 and A 1) 10 mT, was typical of type 2 copper centers. On the addition of nitrite, the EPR features developed spectral features with increased rhombicity, with g 1) 2.29 and A 1) 11 mT, arising from the type 2 catalytic site. As assessed by visible spectroscopy, ferricyanide (E°) +430 mV) was unable to oxidize the H139A enzyme, and this required a 30-fold excess of K 2 IrCl 6 (E°) +867 mV). Oxidation resulted in the EPR spectrum developing additional axial features with g 1) 2.20 and A 1) 9.5 mT, typical of type 1 copper centers. The oxidized enzyme after separation from the excess of K 2 IrCl 6 by gel filtration was a blue-green color with absorbance maxima at 618 and 420 nm. The instability of the protein prevented the precise determination of the midpoint potential, but these properties indicate that it is in the range 700-800 mV, an increase of at least ∼470 mV compared with the native enzyme. This high potential, which is consistent with a trigonal planar geometry of the Cu ion, effectively prevents azurin-mediated electron transfer from the type 1 center to the catalytic type 2 Cu site. However, with dithionite as reductant, 20% of the activity of the wild-type enzyme was observed, indicating that the direct reduction of the catalytic site by dithionite can occur. When CuSO 4 was added to the crude extract before isolation of the enzyme, the Cu content of the purified H139A enzyme increased to 5.7 Cu atoms per trimer. The enzyme remained colorless, and the activity with dithionite as a donor was not significantly increased. The additional copper in such preparations was associated with an axial type 2 Cu EPR signal with g 1) 2.226 and A 1) 18 mT, and which were not changed by the addition of nitrite, consistent with the activity data.
Journal of Molecular Biology, 2006
In Cu-containing nitrite reductase from Alcaligenes faecalis S-6 the axial methionine ligand of the type-1 site was replaced (M150G) to make the copper ion accessible to external ligands that might affect the enzyme's catalytic activity. The type-1 site optical spectrum of M150G (A 460 /A 600 Z 0.71) differs significantly from that of the native nitrite reductase (A 460 / A 600 Z1.3). The midpoint potential of the type-1 site of nitrite reductase M150G (E M Z312(G5) mV versus hydrogen) is higher than that of the native enzyme (E M Z213(G5) mV). M150G has a lower catalytic activity (k cat Z 133(G6) s K1 ) than the wild-type nitrite reductase (k cat Z416(G10) s K1 ). The binding of external ligands to M150G restores spectral properties, midpoint potential (E M !225 mV), and catalytic activity (k cat Z374(G28) s K1 ). Also the M150H (A 460 /A 600 Z7.7, E M Z104(G5) mV, k cat Z0.099(G0.006) s K1 ) and M150T (A 460 /A 600 Z0.085, E M Z340(G5) mV, k cat Z126(G2) s K1 ) variants were characterized. Crystal structures show that the ligands act as allosteric effectors by displacing Met62, which moves to bind to the Cu in the position emptied by the M150G mutation. The reconstituted type-1 site has an otherwise unaltered geometry. The observation that removal of an endogenous ligand can introduce allosteric control in a redox enzyme suggests potential for structural and functional flexibility of coppercontaining redox sites.
Journal of the American Chemical Society, 2007
Copper-containing nitrite reductase harbors a type-1 and a type-2 Cu site. The former acts as the electron acceptor site of the enzyme, and the latter is the site of catalytic action. The effect of the methionine ligand on the reorganization energy of the type-1 site was explored by studying the electrontransfer kinetics between NiR (wild type (wt) and the variants Met150Gly and Met150Thr) with Fe(II)EDTA and Fe(II)HEDTA. The mutations increased the reorganization energy by 0.3 eV (30 kJ mol -1 ). A similar increase was found from pulse radiolysis experiments on the wt NIR and three variants (Met150Gly, Met150His, and Met150Thr). Binding of the nearby Met62 to the type-1 Cu site in Met150Gly (under influence of an allosteric effector) lowered the reorganization energy back to approximately the wt value. According to XRD data the structure of the reduced type-1 site in Met150Gly NiR in the presence of an allosteric effector is similar to that in the reduced wt NiR (solved to 1.85 Å), compatible with the similarity in reorganization energy.
Biochemical Journal, 2001
The blue dissimilatory nitrite reductase (NiR) from Alcaligenes xylosoxidans is a trimer containing two types of Cu centre, three type 1 electron transfer centres and three type 2 centres. The latter have been implicated in the binding and reduction of nitrite. The Cu ion of the type 2 centre of the oxidized enzyme is ligated by three His residues, and additionally has a coordinated water molecule that is also hydrogen-bonded to the carboxyl of Asp*# [Dodd, Van Beeumen, Eady and Hasnain (1998), J. Mol. Biol. 282, 369-382]. Two mutations of this residue have been made, one to a glutamic acid residue and a second to an asparagine residue ; the effects of both mutations on the spectroscopic and catalytic properties of the enzyme have been analysed. EPR spectroscopy revealed that both mutants retained intact type 1 Cu centres with g R l 2.12 (A R l 0 mT) and g U l 2.30 (A U l 6.4 mT), which was consistent with their blue colour, but differed in their activities and in the spectroscopic properties of the type 2 centres. The D92E mutant had an altered geometry of its type 2 centre such that nitrite was no longer capable of binding to elicit changes in the EPR parameters of this centre. Accordingly,
Structures of a Blue-Copper Nitrite Reductase and its Substrate-Bound Complex
Acta Crystallographica Section D-biological Crystallography, 1997
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 of Molecular Biology, 2005
We present high-resolution crystal structures and functional analysis of T1Cu centre mutants of nitrite reductase that perturb the redox potential and the Cys130-His129 "hard-wired" bridge through which electron transfer to the catalytic T2Cu centre occurs. These data provide insight into how activity can be altered through mutational manipulation of the electron delivery centre (T1Cu). The alteration of Cys to Ala results in loss of T1Cu and enzyme inactivation with azurin as electron donor despite the mutant enzyme retaining full nitrite-binding capacity. These data establish unequivocally that no direct transfer of electrons occurs from azurin to the catalytic type 2 Cu centre. The mutation of the axial ligand Met144 to Leu increases both the redox potential and catalytic activity, establishing that the rate-determining step of catalysis is the intermolecular electron transfer from azurin to nitrite reductase.
Inorganic Chemistry, 2004
Copper nitrite reductases contain both an electron-transfer type 1 Cu site and a catalytic type 2 Cu site. We have mutated one of the type 2 copper ligating histidines to observe the effect on catalytic turnover. This mutation has created a unique site where Cu is ligated by 2 His N 2 atoms alone. IC048966P Figure 2. Stereo image (top) of the type 1 and type 2 Cu sites of H129V. The 2Fo-Fc electron density map clearly shows the absence of a Cu-ligating water at the T2Cu site. The superposition (above) of the native protein (red) with H129V (blue) clearly shows the changes that have occurred at the T2Cu site with the mutation of His129. Also, the superposition shows where the waters near the T2Cu in the native protein are and that they are absent in H129V.