Structure of Alcaligenes faecalis nitrite reductase and a copper site mutant, M150E, that contains zinc.
The structures at 2.0 and 2.25 A resolution of native and recombinant nitrite reductase from Alcaligenes faecalis show that they are identical to each other and very similar to nitrite reductase from Achromobacter cycloclastes. The crystallographic structure of a mutant, M150E, which unlike the wild-type protein cannot be reduced by pseudoazurin, shows that the glutamate replacement for methionine binds to a metal at the type I Cu site via only one oxygen. Anomalous scattering data collected at wavelengths of 1.040 and 1.377 A reveal that the metal at the type I site is a Zn. No significant differences from the native structure other than local perturbations at the type I site are seen. A local pseudo 2-fold axis relates the two domains of different monomers which form the active site. The two residues, Asp98 and His255, believed to be involved in catalysis are related by this 2-fold. An unusual (+)-(+) charge interaction between Lys269, Glu279, and His100 helps to orient the active site Cu ligand, His100. A number of negatively charged surface residues create an electrostatic field whose shape suggests that it may serve to direct incoming negatively charged nitrite as well as to dock the electron donor partner, pseudoazurin.
Murphy
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ME
Department of Biological Structure, School of Medicine, University of Washington, Seattle 98195-7420, USA.
Turley
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S
Kukimoto
M
M
Nishiyama
M
M
Horinouchi
S
S
Sasaki
H
H
Tanokura
M
M
Adman
E T
ET
D000421:Alcaligenes / Q000201:enzymology*; D001400:Azurin / Q000031:analogs & derivatives / Q000378:metabolism; D001665:Binding Sites; D003300:Copper / Q000032:analysis*; D018360:Crystallography, X-Ray; D008667:Metalloproteins / Q000737:chemistry*; D008958:Models, Molecular; D009154:Mutation; D009572:Nitrite Reductases / Q000737:chemistry* / Q000235:genetics; D009573:Nitrites / Q000378:metabolism; D011487:Protein Conformation; D011994:Recombinant Proteins / Q000737:chemistry; D015203:Reproducibility of Results; D012542:Scattering, Radiation; D012996:Solutions; D015032:Zinc / Q000032:analysis*
D008667:Metalloproteins; D009573:Nitrites; D011994:Recombinant Proteins; D012996:Solutions; C047163:pseudoazurin; D001400:Azurin; D003300:Copper; D009572:Nitrite Reductases; D015032:Zinc