Laser-flash photolysis indicates that internal electron transfer is triggered by proton uptake by Alcaligenes xylosoxidans copper-dependent nitrite reductase (original) (raw)
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Journal of Biological Chemistry, 2004
The intermolecular electron transfer from Achromobacter cycloclastes pseudoazurin (AcPAZ) to wildtype and mutant Alcaligenes xylosoxidans nitrite reductases (AxNIRs) was investigated using steady-state kinetics and electrochemical methods. The affinity and the electron transfer reaction constant (k ET) are considerably lower between AcPAZ and AxNIR (K m ؍ 1.34 mM and k ET ؍ 0.87 ؋ 10 5 M ؊1 s ؊1) than between AcPAZ and its cognate nitrite reductase (AcNIR) (K m ؍ 20 M and k ET ؍ 7.3 ؋ 10 5 M ؊1 s ؊1). A negatively charged hydrophobic patch, comprising seven acidic residues around the type 1 copper site in AcNIR, is the site of protein-protein interaction with a positively charged hydrophobic patch on AcPAZ. In AxNIR, four of the negatively charged residues (Glu-112, Glu-133, Glu-195, and Asp-199) are conserved at the corresponding positions of AcNIR, whereas the other three residues are not acidic amino acids but neutral amino acids (Ala-83, Ala-191, and Gly-198). Seven mutant AxNIRs with additional negatively charged residues surrounding the hydrophobic patch of AxNIR (A83D, A191E, G198E, A83D/A191E, A93D/G198E, A191E/G198E, and A83D/A191E/G198E) were prepared to enhance the specificity of the electron transport reaction between AcPAZ and AxNIR. The k ET values of these mutants become progressively larger as the number of mutated residues increases. The K m and k ET values of A83D/A191E/G198E (K m ؍ 88 M and k ET ؍ 4.1 ؋ 10 5 M ؊1 s ؊1) are 15-fold smaller and 4.7-fold larger than those of wild-type AxNIR, respectively. These results suggest that the introduction of negatively charged residues into the docking surface of AxNIR facilitates both the formation of electron transport complex and the electron transfer reaction.
Redox-coupled proton transfer mechanism in nitrite reductase revealed by femtosecond crystallography
Proceedings of the National Academy of Sciences, 2016
Significance Copper nitrite reductase (CuNiR) is involved in denitrification of the nitrogen cycle. Synchrotron X-rays rapidly reduce copper sites and decompose the substrate complex structure, which has made crystallographic studies of CuNiR difficult. Using femtosecond X-ray free electron lasers, we determined intact structures of CuNiR with and without nitrite. Based on the obtained structures, we proposed a redox-coupled proton switch model, which provides an explanation for proton-coupled electron transfer (PCET) in CuNiR. PCET is widely distributed through biogenic processes including respiratory and photosynthetic systems and is highly expected to be incorporated into bioinspired molecular devices. Our study also establishes the foundation for future studies on PCET in other systems.
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.
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 The American Chemical Society, 2009
A combination of spectroscopy and DFT calculations has been used to define the geometric and electronic structure of the nitrite bound type 2 (T2) copper site at high and low pH in nitrite reductase from Rhodobacter sphaeroides. At high pH there is no electron transfer from reduced type 1 (T1) to the nitrite bound T2 copper, while protonation triggers T1 → T2 electron transfer and generation of NO. The DFT calculated reaction coordinate for the N-O bond cleavage in nitrite reduction by the reduced T2 copper suggests that the process is best described as proton transfer triggering electron transfer. Bidentate nitrite binding to copper is calculated to play a major role in activating the reductive cleavage of the nitrite bond through backbonding combined with stabilization of the − OH product by coordination to the Cu 2+ .
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.