| 1. |
- Jacobson, Frida, 1975, et al.
(author)
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High resolution X-ray structures of the oxidised and reduced forms of nitrite reductase from Rhodobacter sphaeroides 2.4.3
- 2005
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In: Acta Crystallography D. - 0907-4449 .- 1399-0047. ; 61, s. 1190-1198
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Journal article (peer-reviewed)abstract
- Nitrite reductase is an enzyme operating in the denitrification pathway which catalyses the conversion of nitrite (NO2(-)) to gaseous nitric oxide (NO). Here, crystal structures of the oxidized and reduced forms of the copper-containing nitrite reductase from Rhodobacter sphaeroides 2.4.3 are presented at 1.74 and 1.85 A resolution, respectively. Whereas the structure of the enzyme is very similar to those of other copper-containing nitrite reductases, folding as a trimer and containing two copper sites per monomer, the structures reported here enable conformational differences between the oxidized and reduced forms of the enzyme to be identified. In the type 1 copper site, a rotational perturbation of the side chain of the copper ligand Met182 occurs upon reduction. At the type 2 copper site, a dual conformation of the catalytic residue His287 is observed in the oxidized structure but is lacking in the reduced structure, such that the interactions of the oxidized type 2 copper ion can be regarded as adopting octahedral geometry. These findings shed light on the structural mechanism of the reduction of a copper-bound nitrite to nitric oxide and water.
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| 2. |
- Jacobson, Frida, 1975, et al.
(author)
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pH dependence of copper geometry, reduction potential, and nitrite affinity in nitrite reductase
- 2007
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In: Journal of Biological Chemistry. - 0021-9258 .- 1083-351X. ; 282:9, s. 6347-6355
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Journal article (peer-reviewed)abstract
- 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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