The role of the NADH-dependent nitrite reductase, Nir, from Escherichia coli in fermentative ammonification.

Nitrate and nitrite reduction are of paramount importance for nitrogen assimilation and anaerobic metabolism, and understanding the specific roles of each participating reductase is necessary to describe the biochemical balance that dictates cellular responses to their environments. The soluble, cytoplasmic siroheme NADH-nitrite reductase (Nir) in Escherichia coli is necessary for nitrate/nitrite assimilation but has also been reported to either "detoxify" nitrite, or to carry out fermentative ammonification in support of anaerobic catabolism. Theoretically, nitrite detoxification would be important for anaerobic growth on nitrate, during which excess nitrite would be reduced to ammonium. Fermentative ammonification by Nir would be important for maximization of non-respiratory ATP production during anaerobic growth in the presence of nitrite. Experiments reported here were designed to test the potential role of Nir in fermentative ammonification directly by growing E. coli along with mutant strains lacking Nir or the respiratory nitrite reductase (Nrf) under anaerobic conditions in defined media while monitoring nitrogen utilization and fermentation metabolites. To focus on the role of Nir in fermentative ammonification, pH control was used in most experiments to eliminate nitrite toxicity due to nitric acid formation. Our results demonstrate that Nir confers a significant benefit during fermentative growth that reflects fermentative ammonification rather than detoxification. We conclude that fermentative ammonification by Nir allows for the energetically favorable fermentation of glucose to formate and acetate. These results and conclusions are discussed in light of the roles of Nir in other bacteria and in plants.

Steady-State Kinetics of Phytoglobin-Catalyzed Reduction of Hydroxylamine to Ammonium.

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 SO2•- 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.

Role of Reversible Histidine Coordination in Hydroxylamine Reduction by Plant Hemoglobins (Phytoglobins).

Reduction of hydroxylamine to ammonium by phytoglobin, a plant hexacoordinate hemoglobin, is much faster than that of other hexacoordinate hemoglobins or pentacoordinate hemoglobins such as myoglobin, leghemoglobin, and red blood cell hemoglobin. The reason for differences in reactivity is not known but could be intermolecular electron transfer between protein molecules in support of the required two-electron reduction, hydroxylamine binding, or active site architecture favoring the reaction. Experiments were conducted with phytoglobins from rice, tomato, and soybean along with human neuroglobin and soybean leghemoglobin that reveal hydroxylamine binding as the rate-limiting step. For hexacoordinate hemoglobins, binding is limited by the dissociation rate constant for the distal histidine, while leghemoglobin is limited by an intrinsically low affinity for hydroxylamine. When the distal histidine is removed from rice phytoglobin, a hydroxylamine-bound intermediate is formed and the reaction rate is diminished, indicating that the distal histidine imidazole side chain is critical for the reaction, albeit not for electron transfer but rather for direct interaction with the substrate. Together, these results demonstrate that phytoglobins are superior at hydroxylamine reduction because they have distal histidine coordination affinity constants near 1, and facile rate constants for binding and dissociation of the histidine side chain. Hexacoordinate hemoglobins such as neuroglobin are limited by tighter histidine coordination that blocks hydroxylamine binding, and pentacoordinate hemoglobins have intrinsically lower hydroxylamine affinities.

Electron self-exchange in hemoglobins revealed by deutero-hemin substitution.

Hemoglobins (phytoglobins) from rice plants (nsHb1) and from the cyanobacterium Synechocystis (PCC 6803) (SynHb) can reduce hydroxylamine with two electrons to form ammonium. The reaction requires intermolecular electron transfer between protein molecules, and rapid electron self-exchange might play a role in distinguishing these hemoglobins from others with slower reaction rates, such as myoglobin. A relatively rapid electron self-exchange rate constant has been measured for SynHb by NMR, but the rate constant for myoglobin is equivocal and a value for nsHb1 has not yet been measured. Here we report electron self-exchange rate constants for nsHb1 and Mb as a test of their role in hydroxylamine reduction. These proteins are not suitable for analysis by NMR ZZ exchange, so a method was developed that uses cross-reactions between each hemoglobin and its deutero-hemin substituted counterpart. The resulting electron transfer is between identical proteins with low driving forces and thus closely approximates true electron self-exchange. The reactions can be monitored spectrally due to the distinct spectra of the prosthetic groups, and from this electron self-exchange rate constants of 880 (SynHb), 2900 (nsHb1), and 0.05M(-1) s(-1) (Mb) have been measured for each hemoglobin. Calculations of cross-reactions using these values accurately predict hydroxylamine reduction rates for each protein, suggesting that electron self-exchange plays an important role in the reaction.