Functional Analysis of Conserved Aspartate and Histidine Residues Located Around the Type 2 Copper Site of Copper-Containing Nitri Reductase

Kataoka, Keisuke; Furusawa, Hiroyuki; Takagi, Kohichl; Yamaguchi, Kazuya; Suzuki, Shinnichiro

doi:10.1093/oxfordjournals.jbchem.a022613

Cited by 129 publications

(150 citation statements)

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“…In the deprotonated state the proximal conformation dominates, while protonation allows Asp CAT to adopt the gatekeeper position, independent of the protonation state of His CAT . Experimentally, Asp CAT is in the deprotonated state at pH $6, the optimum pH for CuNiR reduction in Af NiR (Zhang et al, 2000;Kataoka et al, 2000;Kakutani et al, 1981). Protonation of Asp CAT at low pH is consistent with spectroscopic and DFT studies by Ghosh et al (2009).…”

Section: The Protonation State Of Asp Cat Influences the Solvent Accesupporting

confidence: 71%

Active-site protein dynamics and solvent accessibility in nativeAchromobacter cycloclastescopper nitrite reductase

Sen

Horrell

Kekilli

et al. 2017

Int Union Crystallogr J

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Microbial nitrite reductases are denitrifying enzymes that are a major component of the global nitrogen cycle. Multiple structures measured from one crystal (MSOX data) of copper nitrite reductase at 240 K, together with molecular-dynamics simulations, have revealed protein dynamics at the type 2 copper site that are significant for its catalytic properties and for the entry and exit of solvent or ligands to and from the active site. Molecular-dynamics simulations were performed using different protonation states of the key catalytic residues (Asp CAT and His CAT ) involved in the nitrite-reduction mechanism of this enzyme. Taken together, the crystal structures and simulations show that the Asp CAT protonation state strongly influences the active-site solvent accessibility, while the dynamics of the active-site 'capping residue' (Ile CAT ), a determinant of ligand binding, are influenced both by temperature and by the protonation state of Asp CAT . A previously unobserved conformation of Ile CAT is seen in the elevated temperature series compared with 100 K structures. DFT calculations also show that the loss of a bound water ligand at the active site during the MSOX series is consistent with reduction of the type 2 Cu atom.

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Section: The Protonation State Of Asp Cat Influences the Solvent Accesupporting

confidence: 71%

Active-site protein dynamics and solvent accessibility in nativeAchromobacter cycloclastescopper nitrite reductase

Sen

Horrell

Kekilli

et al. 2017

Int Union Crystallogr J

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“…With 10 mM nitrite present, however, the T2D enzyme did show a slight recovery of the AU 595 signal subsequent to reduction, which can be explained by the presence of ϳ10% residual T2Cu typically found in T2D preparations of the enzyme (11,14,37). Alternatively, it has been demonstrated previously that "unmetallated" NiR exhibits some residual catalytic activity (7,16,22,42,43), which may also explain the slight recovery of the AU 595 signal. Because crystal structures did not reveal any interaction of NO 2 Ϫ with the T1Cu center (14,27), the substrate has been suggested to bind to the unmetallated T2Cu center (22).…”

Section: Steady-state Activity Of Axnir In H 2 O and D 2 O-mentioning

confidence: 80%

“…Asp-92 and His-249 have been proposed as acid-base catalysts (18,21,22,28,38), and the abrupt drop in rates at increasing pH may indicate that OH Ϫ can act as a competitive inhibitor for nitrite (39). The relevance of these active site residues, however, as well as the timing of the two protonation steps is still a matter of debate (35,40,41).…”

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confidence: 99%

Demonstration of Proton-coupled Electron Transfer in the Copper-containing Nitrite Reductases

Brenner

Heyes

Hay

et al. 2009

Journal of Biological Chemistry

125

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The reduction of nitrite (NO 2 ؊ ) into nitric oxide (NO), catalyzed by nitrite reductase, is an important reaction in the denitrification pathway. In this study, the catalytic mechanism of the copper-containing nitrite reductase from Alcaligenes xylosoxidans (AxNiR) has been studied using single and multiple turnover experiments at pH 7.0 and is shown to involve two protons. A novel steady-state assay was developed, in which deoxyhemoglobin was employed as an NO scavenger. A moderate solvent kinetic isotope effect (SKIE) of 1.3 ؎ 0.1 indicated the involvement of one protonation to the rate-limiting catalytic step. Laser photoexcitation experiments have been used to obtain single turnover data in H 2 O and D 2 O, which report on steps kinetically linked to inter-copper electron transfer (ET). In the absence of nitrite, a normal SKIE of ϳ1.33 ؎ 0.05 was obtained, suggesting a protonation event that is kinetically linked to ET in substratefree AxNiR. A nitrite titration gave a normal hyperbolic behavior for the deuterated sample. However, in H 2 O an unusual decrease in rate was observed at low nitrite concentrations followed by a subsequent acceleration in rate at nitrite concentrations of >10 mM. As a consequence, the observed ET process was faster in D 2 O than in H 2 O above 0.1 mM nitrite, resulting in an inverted SKIE, which featured a significant dependence on the substrate concentration with a minimum value of ϳ0.61 ؎ 0.02 between 3 and 10 mM. Our work provides the first experimental demonstration of proton-coupled electron transfer in both the resting and substrate-bound AxNiR, and two protons were found to be involved in turnover.

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“…The enzyme receives one electron at the type 1 Cu site from an electron donor protein and catalyzes one electron reduction of NO 2 Ϫ to NO at the type 2 Cu. Moreover, a hydrogen bond network including Asp and His around the type 2 Cu functions as the proton donation to the substrate (2,6,7,9). These active site features have been also observed in the major anaerobically induced outer membrane Cu protein from pathogenic Neisseria gonorrhoeae (10) (AniA).…”

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confidence: 78%

Structure and function of a hexameric copper-containing nitrite reductase

Nojiri

Xie

Inoue

et al. 2007

Proc. Natl. Acad. Sci. U.S.A.

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Dissimilatory nitrite reductase (NIR) is a key enzyme in denitrification, catalyzing the first step that leads to gaseous products (NO, N 2O, and N2). We have determined the crystal structure of a Cu-containing NIR from a methylotrophic denitrifying bacterium, Hyphomicrobium denitrificans, at 2.2-Å resolution. The overall structure of this H. denitrificans NIR reveals a trigonal prism-shaped molecule in which a monomer consisting of 447 residues and three Cu atoms is organized into a unique hexamer (i.e., a tightly associated dimer of trimers). Each monomer is composed of an N-terminal region containing a Greek key ␤-barrel folding domain, cupredoxin domain I, and a C-terminal region containing cupredoxin domains II and III. Both cupredoxin domains I and II bind one type 1 Cu and are combined with a long loop comprising 31 amino acid residues. The type 2 Cu is ligated at the interface between domain II of one monomer and domain III of an adjacent monomer. Between the two trimeric C-terminal regions are three interfaces formed by an interaction between the domains I, and the type 1 Cu in the domain is required for dimerization of the trimer. The type 1 Cu in domain II functions as an electron acceptor from an electron donor protein and then transfers an electron to the type 2 Cu, binding the substrate to reduce nitrite to NO. The discussion of the intermolecular electron transfer process from cytochrome c 550 to the H. denitrificans NIR is based on x-ray crystallographic and kinetic results.denitrification ͉ electron transfer ͉ redox partner ͉ intermolecular interaction ͉ cupredoxin

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Functional Analysis of Conserved Aspartate and Histidine Residues Located Around the Type 2 Copper Site of Copper-Containing Nitri Reductase

Cited by 129 publications

References 0 publications

Active-site protein dynamics and solvent accessibility in nativeAchromobacter cycloclastescopper nitrite reductase

Active-site protein dynamics and solvent accessibility in nativeAchromobacter cycloclastescopper nitrite reductase

Demonstration of Proton-coupled Electron Transfer in the Copper-containing Nitrite Reductases

Structure and function of a hexameric copper-containing nitrite reductase

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