Cyanide binding to cd(1) nitrite reductase from Pseudomonas aeruginosa: role of the active-site His369 in ligand stabilization.
Cyanide binding to fully reduced Pseudomonas aeruginosa cd(1) nitrite reductase (Pa cd(1) NiR) has been investigated for the wild-type enzyme and a site-directed mutant in which the active-site His369 was replaced by Ala. This mutation reduces the affinity toward cyanide (by approximately 13-fold) and especially decreases the rate of binding of cyanide to the reduced d(1) heme (by approximately 100-fold). The crystal structure of wild-type reduced Pa cd(1) NiR saturated with cyanide was determined to a resolution of 2.7 A. Cyanide binds to the iron of the d(1) heme, with an Fe-C-N angle of 168 degrees for both subunits of the dimer and only His369 is within hydrogen bonding distance of the nitrogen atom of the ligand. These results suggest that in Pa cd(1) NiR the invariant distal residue His369 plays a dominant role in controlling the binding of anionic ligands and allow the discussion of the mechanism of cyanide binding to the wild-type enzyme.
Sun
Wenliang
W
Dipartimento di Scienze Biochimiche A. Rossi Fanelli and Centro di Biologia Molecolare del CNR, Università di Roma La Sapienza, Rome, Italy.
Arese
Marzia
M
Brunori
Maurizio
M
Nurizzo
Didier
D
Brown
Kieron
K
Cambillau
Christian
C
Tegoni
Mariella
M
Cutruzzolà
Francesca
F
D019943:Amino Acid Substitution; D001665:Binding Sites / Q000502:physiology; D003486:Cyanides / Q000737:chemistry*; D008024:Ligands; D008958:Models, Molecular; D008969:Molecular Sequence Data; D016297:Mutagenesis, Site-Directed; D009572:Nitrite Reductases / Q000737:chemistry* / Q000235:genetics; D017434:Protein Structure, Tertiary; D011550:Pseudomonas aeruginosa / Q000201:enzymology*; D013053:Spectrophotometry; D014961:X-Ray Diffraction
D003486:Cyanides; D008024:Ligands; D009572:Nitrite Reductases