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 dissimilator y denitrification is catalyzed by the enzyme nitrite reductase (NiR), a homodimer containing a covalently bound c heme and a d(1) heme per s ubunit, We report the purification and characterization of the first s ingle mutant of P. aeruginosa cd(1) NiR in which Tyr(10) has been repl aced by Phe; this amino acid mas chosen as a possibly important residu e in the catalytic mechanism of this enzyme based on the proposal (Ful op, V., Moir, J.W.B., Ferguson, S.J. and Hajdu, J. (1995) Cell 81, 369 -377) that the topologically homologous Tyr(25) plays a crucial role i n controlling the activity of the cd(1) NiR from Thiosphaera pantotrop ha. Our results show that in P. aeruginosa NiR substitution of Tyr(10) with Phe has no effect on the activity, optical spectroscopy and elec tron transfer kinetics of the enzyme, indicating that distal coordinat ion of the Fe3+ of the d(1) heme is provided by different side-chains in different species. (C) 1997 Federation of European Biochemical Soci eties.