The long-term aim of this project is to understand the regulation of heme biosynthesis in the symbiotic nitrogen-fixing bacterium Bradyrhizobium japonicum. The iron-containing porphyrin ring (heme) is involved in several enzymatic reactions requiring microanaerobic conditions necessary for efficient nitrogen fixation (free-living or symbiotic). The multienzyme pathway for heme formation ends with the insertion of iron into the protoporphyrin ring in a reaction catalyzed by ferrochelatase. Because iron may be a limiting nutrient and porphyrins are cytotoxic, this suggests the need for coordination of heme biosynthesis with iron availability. A novel protein called Irr has been recently identified and proposed to mediate iron control of heme biosynthesis. Irr is present under iron limitation and negatively regulates the heme biosynthesis gene hemB, preventing protoporphyrin accumulation from exceeding iron availability. Irr is a conditionally stable protein that degrades rapidly when cells are exposed to iron. Heme is an effector molecule in this iron-mediated degradation, binding to a specific motif in Irr. Irr also binds ferrochelatase directly. This proposal will test the hypothesis that Irr mediates iron control of the heme pathway by responding to the status of protoporphyrin and heme at the site of heme synthesis. The role of heme in mediating iron-dependent Irr degradation will be elucidated. Specifically, two plausible mechanisms will be tested: heme catalysis of Irr oxidation leading to degradation, or heme-induced conformational change in Irr, rendering it accessible to proteases. The binding of ferrochelatase to Irr and the effect of this binding on Irr activity will also be studied. This work focuses on a metabolic pathway that is important for energy production and its novel regulation by bacterial cells, and thus should provide insights on the ability of bacteria to adjust to diverse environmental conditions. The environmental adaptability of Bradyrhizobium japonicum is of special interest because this bacterium establishes a nitrogen-fixing symbiotic association with soybeans. Because this relationship is beneficial to a commercially important plant crop, B. japonicum is thus of economic and agricultural interest. These studies will also involve the training of graduate and undergraduate students, including women and underrepresented minority trainees.
