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We report the purification and characterization of the first single mutant of P. aeruginosa cd₁ NiR in which Tyr¹⁰ has been replaced by Phe; this amino acid was chosen as a possibly important residue in the catalytic mechanism of this enzyme based on the proposal (Fülöp, V., Moir, J.W.B.,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.