Control of catalysis in globin coupled adenylate cyclase by a globin-B domain

• HemAC-Lm has two globin domains (globin-A and globin-B).
• Heme can only bind at globin-A domain but not at globin-B domain in HemAC-Lm.
• In absence of globin-A domain, globin-B domain inhibits AC activity.
• Globin-A domain relieves globin-B domain induced inhibition in HemAC-Lm.

The globin coupled heme containing adenylate cyclase from Leishmania major (HemAC-Lm) has two globin domains (globin-A and globin-B). Globin-B domain (210–360 amino acids) may guide the interaction between globin-A and adenylate cyclase domains for the regulation of catalysis. We investigated the role of globin-B domain in HemAC-Lm by constructing a series of mutants namely Δ209 (209 amino acids deleted), Δ360 (360 amino acids deleted), H161A, H311A and H311A-Δ209. Spectroscopic data suggest that the Δ209 and H311A-Δ209 proteins to be Fe2+–O2 form and apo form, respectively, indicating that His311 residue in the globin-B domain is crucial for heme binding in Δ209 protein. However, the H311A mutant is still of the Fe2+–O2 form whereas H161A mutant shows the apo form, indicating that only His161 residue in the globin-A domain is responsible for heme binding in full length enzyme. cAMP measurements suggest that the activities of Δ360 and Δ209 proteins were ∼10 and ∼1000 times lesser than full length enzyme, respectively, leading to the fact that globin-B domain inhibited catalysis rather than activation in absence of globin-A domain. These data suggest that the O2 bound globin-A domain in HemAC-Lm allows the best cooperation of the catalytic domain interactions to generate optimum cAMP.