Effect of the Methionine Ligand on the Reorganization Energy of the Type-1 Copper Site of Nitrite Reductase (original) (raw)
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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 Biological Chemistry, 2006
The homotrimeric copper-containing nitrite reductase (NiR) contains one type-1 and one type-2 copper center per monomer. Electrons enter through the type-1 site and are shuttled to the type-2 site where nitrite is reduced to nitric oxide. To investigate the catalytic mechanism of NiR the effects of pH and nitrite on the turnover rate in the presence of three different electron donors at saturating concentrations were measured. The activity of NiR was also measured electrochemically by exploiting direct electron transfer to the enzyme immobilized on a graphite rotating disk electrode. In all cases, the steady-state kinetics fitted excellently to a randomsequential mechanism in which electron transfer from the type-1 to the type-2 site is rate-limiting. At low [NO 2 ؊ ] reduction of the type-2 site precedes nitrite binding, at high [NO 2 ؊ ] the reverse occurs. Below pH 6.5, the catalytic activity diminished at higher nitrite concentrations, in agreement with electron transfer being slower to the nitrite-bound type-2 site than to the water-bound type-2 site. Above pH 6.5, substrate activation is observed, in agreement with electron transfer to the nitrite-bound type-2 site being faster than electron transfer to the hydroxyl-bound type-2 site. To study the effect of slower electron transfer between the type-1 and type-2 site, NiR M150T was used. It has a type-1 site with a 125-mV higher midpoint potential and a 0.3-eV higher reorganization energy leading to an ϳ50-fold slower intramolecular electron transfer to the type-2 site. The results confirm that NiR employs a random-sequential mechanism. Copper-containing nitrite reductase (NiR) 3 is one of the enzymes of the denitrification pathway (1). Denitrification globally recycles fixed nitrogen (NO 3 Ϫ , NO 2 Ϫ) to the atmosphere (N 2) with NO and N 2 O being
Biochimica et biophysica acta, 2017
The Cys-His bridge as electron transfer conduit in the enzymatic catalysis of nitrite to nitric oxide by nitrite reductase from Sinorhizobium meliloti 2011 (SmNir) was evaluated by site-directed mutagenesis, steady state kinetic studies, UV-vis and EPR spectroscopic measurements as well as computational calculations. The kinetic, structural and spectroscopic properties of the His171Asp (H171D) and Cys172Asp (C172D) SmNir variants were compared with the wild type enzyme. Molecular properties of H171D and C172D indicate that these point mutations have not visible effects on the quaternary structure of SmNir. Both variants are catalytically incompetent using the physiological electron donor pseudoazurin, though C172D presents catalytic activity with the artificial electron donor methyl viologen (kcat=3.9(4) s(-1)) lower than that of wt SmNir (kcat=240(50) s(-1)). QM/MM calculations indicate that the lack of activity of H171D may be ascribed to the N(δ1)H…OC hydrogen bond that partially...
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.
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,
FEBS Letters, 1998
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 k i = k I3P +k P3I of 450 þ 30 s 3I 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 I3P = 185 þ 12 s 3I and k P3I 265 þ 18 s 3I. The temperature dependence of K allowed us to determine the v vH³ value of the ET equilibrium to be 12.1 kJ mol 3I. Measurements of the temperature dependence of the ET process yielded the following activation parameters: forward reaction, v vH g = 22.7 þ 3.4 kJ mol 3I and v vS g = 3126 þ 11 J K 3I mol 3I ; backward reaction, v vH g = 10.6 þ 1.7 kJ mol 3I and v vS g = 3164 þ 15 J K 3I mol 3I. X-ray crystallographic studies of NiRs suggest that the most probable ET pathway linking the two copper sites consists of Cys IQT , which provides the thiolate ligand to the type 1 copper ion, and the adjacent His IQS 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.
Impact of residues remote from the catalytic centre on enzyme catalysis of copper nitrite reductase
Nature Communications, 2014
Enzyme mechanisms are often probed by structure-informed point mutations and measurement of their effects on enzymatic properties to test mechanistic hypotheses. In many cases, the challenge is to report on complex, often inter-linked elements of catalysis. Evidence for long-range effects on enzyme mechanism resulting from mutations remains sparse, limiting the design/redesign of synthetic catalysts in a predictable way. Here we show that improving the accessibility of the active site pocket of copper nitrite reductase by mutation of a surface-exposed phenylalanine residue (Phe306), located 12 Å away from the catalytic site type-2 Cu (T2Cu), profoundly affects intra-molecular electron transfer, substrate-binding and catalytic activity. Structures and kinetic studies provide an explanation for the lower affinity for the substrate and the alteration of the rate-limiting step in the reaction. Our results demonstrate that distant residues remote from the active site can have marked effects on enzyme catalysis, by driving mechanistic change through relatively minor structural perturbations. NO 2 , nitrite; r.m.s.d., root mean square deviation. *(F306C -no Ramachandran outliers, for 660 residues 97.92% favoured 2.08% allowed; F306C þ NO 2 -no Ramachandran outliers, for 331 residues 96.78% favoured, 3.22% allowed). | www.nature.com/naturecommunications NO 2 , nitrite; r.m.s.d., root mean square deviation; T1Cu, type-1 Cu; T2Cu, type-2 Cu; WT, wild type. *Values are taken from ref. 7. Values shown for T1Cu and T2Cu at pH 7 are obtained after fitting the data sets from a single experiment to the Nernst equation. Since the T1Cu reduction potential is pH independent 16 , the values for T2Cu at pH 5.8 were calculated from the relative absorbance recovery in the flash-photolysis experiments performed at pH 5.8.
Biochemistry, 2005
A pulsed electron paramagnetic resonance study has been performed on the type 2 copper site of nitrite reductase (NiR) from Alcaligenes faecalis. The H145A mutant, in which histidine 145 is replaced by alanine, was studied by ESEEM and HYSCORE experiments at 9 GHz on frozen solutions. This mutant contains a reduced type 1 copper site which allowed a selective investigation of the type 2 site of H145A and of its nitrite-bound form H145A (NO 2 -). The experiments yielded hyperfine and quadrupole parameters of the remote nitrogens of two of the histidines in the type 2 copper site of the protein and revealed the changes of these values induced by substrate binding ( 14 NO 2and 15 NO 2 -). The HYSCORE experiments displayed a signal of 15 NO 2bound to H145A, from which hyperfine parameters of the nitrite nitrogen were estimated. The small isotropic hyperfine coupling, 0.36 MHz, of the nitrite nitrogen ( 14 N) suggests that the substrate binds in an axial position to the copper in the type 2 site and that the molecular orbital containing the unpaired electron extends onto the substrate. This and other changes in the EPR parameters occurring after nitrite binding suggest a change in electronic structure of the site, which most likely prepares the site for the catalytic reaction. We propose that this change is essential for the reaction to occur.