RESUMO
Phytoglobins are plant hexacoordinate hemoglobins with reversible coordination of a histidine side chain to the ligand binding site of the heme iron. They mediate electron transfer reactions such as nitric oxide scavenging and are particularly efficient at reducing nitrite and hydroxylamine. Previous work with phytoglobins has focused only on single turnovers of these reactions and has not revealed whether structural features, such as histidine hexacoordination, play a prominent role in the complete catalytic cycle. This work characterizes steady-state phytoglobin catalysis of reduction of hydroxylamine to ammonium using two different chemical reductants. Km and kcat values were measured for rice phytoglobin, horse myoglobin, human neuroglobin, and a rice phytoglobin mutant protein in which the hexacoordinating histidine has been replaced with leucine (Phyt:H73L). The results demonstrate that phytoglobin catalysis driven by benzyl viologen is limited only by the dissociation rate constant for the distal histidine. This is consistent with the rate limit in single-turnover experiments and demonstrates that the kinetics of hydroxylamine binding, and not phytoglobin reduction, ultimately governs the reaction. Catalysis by the other proteins that either lack or have tighter hexacoordination is much slower, suggesting that facile reversibility of the bond between the distal histidine and the heme iron is needed to allow both substrate binding and heme iron reduction. On the other hand, catalysis driven by dithionite is limited by SO2â¢- concentrations and is similar for all of these proteins, suggesting that dithionite is not a good reducing agent for evaluation of the catalytic properties of hemoglobins.
