Reaction of N-hydroxyguanidine with the ferrous–oxy state of a heme peroxidase cavity mutant: A model for the reactions of nitric oxide synthase

Abstract

Yeast cytochrome c peroxidase was used to construct a model for the reactions catalyzed by the second cycle of nitric oxide synthase. The R48A/W191F mutant introduced a binding site for N-hydroxyguanidine near the distal heme face and removed the redox active Trp-191 radical site. Both the R48A and R48A/W191F mutants catalyzed the H 2O 2 dependent conversion of N-hydroxyguanidine to N-nitrosoguanidine. It is proposed that these reactions proceed by direct one-electron oxidation of NHG by the Fe +4 O center of either Compound I (Fe +4 O, porph + ) or Compound ES (Fe +4 O, Trp + ). R48A/W191F formed a Fe +2O 2 complex upon photolysis of Fe +2CO in the presence of O 2, and N-hydroxyguanidine was observed to react with this species to produce products, distinct from N-nitrosoguanidine, that gave a positive Griess reaction for nitrate + nitrite, a positive Berthelot reaction for urea, and no evidence for formation of NO . It is proposed that HNO and urea are produced in analogy with reactions of nitric oxide synthase in the pterin-free state.