Mutagenesis of nitrite reductase from Pseudomonas aeruginosa: tyrosine-10 in the c heme domain is not involved in catalysis.
In Pseudomonas aeruginosa, conversion of nitrite to NO in dissimilatory denitrification is catalyzed by the enzyme nitrite reductase (NiR), a homodimer containing a covalently bound c heme and a d1 heme per subunit. We report the purification and characterization of the first single mutant of P. aeruginosa cd1 NiR in which Tyr10 has been replaced by Phe; this amino acid was chosen as a possibly important residue in the catalytic mechanism of this enzyme based on the proposal (Fulop, V., Moir, J.W.B., Ferguson, S.J. and Hajdu, J. (1995) Cell 81, 369-377) that the topologically homologous Tyr25 plays a crucial role in controlling the activity of the cd1 NiR from Thiosphaera pantotropha. Our results show that in P. aeruginosa NiR substitution of Tyr10 with Phe has no effect on the activity, optical spectroscopy and electron transfer kinetics of the enzyme, indicating that distal coordination of the Fe3+ of the d1 heme is provided by different side-chains in different species.
Cutruzzolà
F
F
Dipartimento di Scienze Biochimiche A. Rossi Fanelli and Centro di Biologia Molecolare del CNR, Università di Roma La Sapienza, Italy. cutruzzola@axcasp.caspur.it
Arese
M
M
Grasso
S
S
Bellelli
A
A
Brunori
M
M
D000595:Amino Acid Sequence; D002384:Catalysis; D006418:Heme / Q000378:metabolism; D007700:Kinetics; D008969:Molecular Sequence Data; D016297:Mutagenesis, Site-Directed; D009572:Nitrite Reductases / Q000235:genetics / Q000378:metabolism*; D011550:Pseudomonas aeruginosa / Q000201:enzymology / Q000235:genetics*; D016958:Pseudomonas putida / Q000235:genetics; D017386:Sequence Homology, Amino Acid; D014443:Tyrosine / Q000235:genetics / Q000378:metabolism*
D014443:Tyrosine; D006418:Heme; D009572:Nitrite Reductases