Catalytic Roles for Two Water Bridged Residues (Asp-98 and His-255) in the Active Site of Copper-containing Nitrite Reductase

Boulanger, Martin J.; Kukimoto, Mutsuko; Nishiyama, Makoto; Horinouchi, Sueharu; Murphy, M.E.P.

doi:10.1074/jbc.m001859200

Cited by 128 publications

(163 citation statements)

References 35 publications

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“…Extensive spectroscopic and structural studies of CuNiRs (Strange et al, 1999;Boulanger et al, 2000;Boulanger & Murphy, 2001;Tocheva et al, 2004Tocheva et al, , 2007Antonyuk et al, 2005;Krzemiń ski et al, 2011;Wijma et al, 2006;Hough et al, 2008;Leferink et al, 2011Leferink et al, , 2012 have suggested a general catalytic mechanism in which bound water in the resting state is replaced by nitrite, and internal electron transfer occurs between the T1Cu and T2Cu sites. Two protons are required for the reaction, which are thought to be provided by Asp98 (Asp CAT ) and His255 (His CAT ) via a bridging water molecule, leading to cleavage of the bound NO 2 À to produce NO and H 2 O. Conformational rearrangements of Asp CAT have been proposed to be important in substrate guidance and proton transfer (Antonyuk et al, 2005), while His CAT may respond to the redox state.…”

Section: Introductionmentioning

confidence: 99%

Enzyme catalysis captured using multiple structures from one crystal at varying temperatures

Horrell

Kekilli

Sen

et al. 2018

IUCrJ

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PDB references: copper nitrite reductase, 190 K data set 1, 5of5; 190 K data set 2, 5of6; 190 K data set 10, 5of7; 190 K data set 18, 5of8; 190 K data set 50, 5ofc; 190 K data set 69, 5ofd; 190 K data set 75, 5ofe; RT data set 1, 5off; RT data set 2, 5ofg; RT data set 3, 5ofh; RT data set 4, 5og2; RT data set 5, 5og3; RT data set 6, 5og4; RT data set 7, 5og5; RT data set 8, 5og6; RT data set 9, 5ogf; RT data set 10, 5ogg High-resolution crystal structures of enzymes in relevant redox states have transformed our understanding of enzyme catalysis. Recent developments have demonstrated that X-rays can be used, via the generation of solvated electrons, to drive reactions in crystals at cryogenic temperatures (100 K) to generate 'structural movies' of enzyme reactions. However, a serious limitation at these temperatures is that protein conformational motion can be significantly supressed. Here, the recently developed MSOX (multiple serial structures from one crystal) approach has been applied to nitrite-bound copper nitrite reductase at room temperature and at 190 K, close to the glass transition. During both series of multiple structures, nitrite was initially observed in a 'top-hat' geometry, which was rapidly transformed to a 'side-on' configuration before conversion to side-on NO, followed by dissociation of NO and substitution by water to reform the resting state. Density functional theory calculations indicate that the top-hat orientation corresponds to the oxidized type 2 copper site, while the side-on orientation is consistent with the reduced state. It is demonstrated that substrate-to-product conversion within the crystal occurs at a lower radiation dose at 190 K, allowing more of the enzyme catalytic cycle to be captured at high resolution than in the previous 100 K experiment. At room temperature the reaction was very rapid, but it remained possible to generate and characterize several structural states. These experiments open up the possibility of obtaining MSOX structural movies at multiple temperatures (MSOX-VT), providing an unparallelled level of structural information during catalysis for redox enzymes.

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Section: Introductionmentioning

confidence: 99%

Enzyme catalysis captured using multiple structures from one crystal at varying temperatures

Horrell

Kekilli

Sen

et al. 2018

IUCrJ

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“…The normal catalytic product, NO, is thought to exit through these same channels. An isoleucine residue (Ile CAT ) that caps the active-site pocket has been proposed to provide steric constraints to ligand access and selectivity to ligand binding (Boulanger & Murphy, 2003;Tocheva et al, 2008), while critical aspartate (Asp CAT ) and histidine (His CAT ) residues are required for the correct ligand positioning, hydrogen bonding and proton delivery during catalysis (Antonyuk et al, 2005;Boulanger et al, 2000;Boulanger & Murphy, 2001). In Achromobacter cycloclastes nitrite reductase (AcNiR) these are the Ile257, Asp98 and His255 residues.…”

Section: Introductionmentioning

confidence: 99%

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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“…X-ray crystal structures have been determined for the green NiRs from Achromobacter cycloclastes (AcNiR) (9,10) and Alcaligenes faecalis NiR (4,(11)(12)(13) and the blue Alcaligenes xylosoxidans (AxNiR) (14,15), for which an atomic resolution structure has been reported recently (5). For the green nitrite reductases, resolution of the structure has steadily improved with the highest resolution being 1.4 Å.…”

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confidence: 99%

“…The type 2 Cu ion (T2Cu) has (His) 3 -H 2 O (or OH Ϫ ) ligation with one of the His residues being provided by an adjacent monomer. Mutagenesis studies have shown that the active site pocket also has conserved aspartyl (Asp-98) and histidinyl (His-255) residues that are essential for enzymatic activity (4)(5)(6)(7) and an isoleucine (Ile-257) residue that is important for optimum positioning of nitrite before catalysis (8). The residues His-255 and Ile-257 are provided by the adjacent monomer.…”

mentioning

confidence: 99%

Atomic resolution structures of resting-state, substrate- and product-complexed Cu-nitrite reductase provide insight into catalytic mechanism

Antonyuk

Strange

Sawers

et al. 2005

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

174

258

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Copper-containing nitrite reductases catalyze the reduction of nitrite to nitric oxide (NO), a key step in denitrification that results in the loss of terrestrial nitrogen to the atmosphere. They are found in a wide variety of denitrifying bacteria and fungi of different physiology from a range of soil and aquatic ecosystems. Structural analysis of potential intermediates in the catalytic cycle is an important goal in understanding enzyme mechanism. Using ''crystal harvesting'' and substrate-soaking techniques, we have determined atomic resolution structures of four forms of the green Cu-nitrite reductase, from the soil bacterium Achromobacter cycloclastes. These structures are the resting state of the enzyme at 0.9 Å, two species exhibiting different conformations of nitrite bound at the catalytic type 2 Cu, one of which is stable and also has NO present, at 1.10 Å and 1.15 Å, and a stable form with the product NO bound side-on to the catalytic type 2 Cu, at 1.12 Å resolution. These structures provide incisive insights into the initial binding of substrate, its repositioning before catalysis, bond breakage (O-NO), and the formation of a stable NO adduct.catalysis ͉ denitrification ͉ enzyme mechanism ͉ nitrite and nitric oxide binding ͉ crystal structures

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Catalytic Roles for Two Water Bridged Residues (Asp-98 and His-255) in the Active Site of Copper-containing Nitrite Reductase

Cited by 128 publications

References 35 publications

Enzyme catalysis captured using multiple structures from one crystal at varying temperatures

Enzyme catalysis captured using multiple structures from one crystal at varying temperatures

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

Atomic resolution structures of resting-state, substrate- and product-complexed Cu-nitrite reductase provide insight into catalytic mechanism

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