Phytoglobins are hexa-coordinated hemoglobins found in plants, whose physiological role is ambiguous. Phytoglobins are known to catalyze the reduction of inorganic nitrogenous compounds such as hydroxylamine. This reduction reaction has been studied extensively by single turnover reactions. However, currently there is no information available on the multi-turnover reactions catalyzed by phtyoglobins in presence of exogenous electron source. In this study we present the results of using of dithionite as an external source of electron for the reduction of hydroxylamine, which gives a maximal rate of 2.8s-1. Furthermore, we demonstrate that the rate-limiting step in this case is the electron transfer from dithionite to the substrate bound phytoglobin. This completely contrasts with what was observed previously in the single turnover assay.

Hydroxylamine reduction is the only known 2-electron reduction process catalyzed by phytoglobin to date. In the current study we also present a novel 2-electron reduction of hydrazine to ammonia catalyzed by phytoglobin. Hydrazine is an intermediate produced during the reduction of nitrogen to ammonia by nitrogenase enzyme. Hence, this discovery opens avenues for engineering phytoglobins for catalyzing the production of ammonia from dinitrogen. Interestingly, this reaction is also limited by electron transfer rate of the dithionite to hydrazine bound phytoglobins. Therefore catalytic rate of ammonia production by phtyoglobins can be improved by introducing a more robust reducing system. We believe this can be achieved by using physiological proteins responsible for reduction of phytoglobins. Hence, this presents a need for characterization and optimization of the physiological proteins responsible for phytoglobin reduction.