Side-On Copper-Nitrosyl Coordination by Nitrite Reductase

Tocheva, Elitza I.; Rosell, Federico I.; Mauk, A. Grant; Murphy, M.E.P.

doi:10.1126/science.1095109

Cited by 221 publications

(341 citation statements)

References 39 publications

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“…Surprisingly, in our first structure (1.55-Å resolution; data not shown) of the ''as-isolated'' AcNiR we found that the T2Cu site had NO bound in a side-on manner, similar to that reported recently for crystals of reduced Alcaligenes faecalis NiR exposed to NO under anaerobic conditions (20). Because we had not added exogenous NO, and because the crystals or protein had not been reduced, we decided to look at further crystals from the same bulk culture.…”

Section: Resultssupporting

confidence: 55%

“…These studies have been in general agreement that nitrite binds to the T2Cu ion via O in an asymmetric bidentate fashion. Recently, Tocheva et al (20) have noted a face-on interaction of the nitrite with Cu in their most recent 1.4-Å resolution structure compared with previous observations of bent O coordination. An examination of the Protein Data Bank (21) reveals a significant variation in the position and orientation of nitrite in the nitrite-bound CuNiRs.…”

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confidence: 99%

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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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Section: Resultssupporting

confidence: 55%

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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“…For example, a membrane-associated nitrite reductase metabolon with deoxyHb and metHb, anion exchange protein, carbonic anhydrase, aquaporin, and rhesus channels could bring together nitrite, proton, deoxyheme, and highly hydrophobic channels that could serve to concentrate the lipophilic NO at the membrane complex (52). Indeed, the heme pocket distal histidine may donate a proton to the reaction, an effect analogous to the proton donation by aspartate in the bacterial nitrite reductase enzyme (53). This histidine proton donation to nitrite would serve to both position and stabilize nitrite in the heme pocket and accelerate the reaction by forming nitrous acid.…”

Section: Discussionmentioning

confidence: 99%

Enzymatic function of hemoglobin as a nitrite reductase that produces NO under allosteric control

Huang¹

2005

Journal of Clinical Investigation

472

511

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Hypoxic vasodilation is a fundamental, highly conserved physiological response that requires oxygen and/or pH sensing coupled to vasodilation. While this process was first characterized more than 80 years ago, the precise identity and mechanism of the oxygen sensor and mediators of vasodilation remain uncertain. In support of a possible role for hemoglobin (Hb) as a sensor and effector of hypoxic vasodilation, here we show biochemical evidence that Hb exhibits enzymatic behavior as a nitrite reductase, with maximal NO generation rates occurring near the oxy-to-deoxy (R-to-T) allosteric structural transition of the protein. The observed rate of nitrite reduction by Hb deviates from second-order kinetics, and sigmoidal reaction progress is determined by a balance between 2 opposing chemistries of the heme in the R (oxygenated conformation) and T (deoxygenated conformation) allosteric quaternary structures of the Hb tetramer -the greater reductive potential of deoxyheme in the R state tetramer and the number of unligated deoxyheme sites necessary for nitrite binding, which are more plentiful in the T state tetramer. These opposing chemistries result in a maximal nitrite reduction rate when Hb is 40-60% saturated with oxygen (near the Hb P 50 ), an apparent ideal set point for hypoxiaresponsive NO generation. These data suggest that the oxygen sensor for hypoxic vasodilation is determined by Hb oxygen saturation and quaternary structure and that the nitrite reductase activity of Hb generates NO gas under allosteric and pH control.

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“…The atomic coordinates and structure factors (code 2DWS,2DY2,and 2DWT) have been argued that it is actually the protonated HNO 2 species that binds to the active site (16,17). Recent studies have shown that before the NO (g) product leaves the type 2 copper site, it is bound to the copper ion as a side-on copper-nitrosyl intermediate (17,18). Near the type 2 site there are also the catalytic residues Asp-129 and His-287, which have been found to be crucial to enzyme activity and nitrite binding (9, 16, 19 -22).…”

mentioning

confidence: 99%

pH Dependence of Copper Geometry, Reduction Potential, and Nitrite Affinity in Nitrite Reductase

Jacobson

Pistorius

Farkas

et al. 2007

Journal of Biological Chemistry

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Many properties of copper-containing nitrite reductase are pH-dependent, such as gene expression, enzyme activity, and substrate affinity. Here we use x-ray diffraction to investigate the structural basis for the pH dependence of activity and nitrite affinity by examining the type 2 copper site and its immediate surroundings in nitrite reductase from Rhodobacter sphaeroides 2.4.3. At active pH the geometry of the substrate-free oxidized type 2 copper site shows a near perfect tetrahedral geometry as defined by the positions of its ligands. At higher pH values the most favorable copper site geometry is altered toward a more distorted tetrahedral geometry whereby the solvent ligand adopts a position opposite to that of the His-131 ligand. This pH-dependent variation in type 2 copper site geometry is discussed in light of recent computational results. When co-crystallized with substrate, nitrite is seen to bind in a bidentate fashion with its two oxygen atoms ligating the type 2 copper, overlapping with the positions occupied by the solvent ligand in the high and low pH structures. Fourier transformation infrared spectroscopy is used to assign the pH dependence of the binding of nitrite to the active site, and EPR spectroscopy is used to characterize the pH dependence of the reduction potential of the type 2 copper site. Taken together, these spectroscopic and structural observations help to explain the pH dependence of nitrite reductase, highlighting the subtle relationship between copper site geometry, nitrite affinity, and enzyme activity.

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Side-On Copper-Nitrosyl Coordination by Nitrite Reductase

Cited by 221 publications

References 39 publications

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

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

Enzymatic function of hemoglobin as a nitrite reductase that produces NO under allosteric control

pH Dependence of Copper Geometry, Reduction Potential, and Nitrite Affinity in Nitrite Reductase

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