title: The ferrous-dioxy complex of Leishmania major globin coupled heme containing adenylate cyclase: the role of proximal histidine on its stability.
abstract: Recently we have described the globin-coupled heme containing adenylate cyclase from Leishmania major (HemAC-Lm) that shows an O2 dependent cAMP signaling (Sen Santara, et. al. Proc. Natl. Acad. Sci. U.S.A. 110, 16790-16795 (2013)). The heme iron of HemAC-Lm is expected to participate in oxygen binding and activates adenylate cyclase activity during catalysis, but its interactions with O2 are uncharacterized. We have utilized the HemAC-Lm and stopped-flow methods to study the formation and decay of the HemAC-Lm oxygenated complex at 25°C. Mixing of the ferrous HemAC-Lm with air-saturated buffer generates a very stable oxygenated complex with absorption maxima at 414, 540 and 576nm. The distal axial ligand in the deoxygenated ferrous HemAC-Lm is displaced by O2 at a rate of ~10s(-1). To prepare apoprotein of heme iron in HemAC-Lm, we have mutated the proximal His161 to Ala and characterized the mutant protein. The apo as well as heme reconstituted ferric state of the mutant protein shows a ~30 fold lower catalytic activity compared to oxygenated form of wild type protein. The oxygenated form of heme reconstituted mutant protein is highly unstable (decay rate=6.1s(-1)). Decomposition of the oxygenated intermediate is independent of O2 concentration and is monophasic. Thus, the stabilization of ferrous-oxy species is an essential requirement in the wild type HemAC-Lm for a conformational alteration in the sensor domain that, sequentially, activates the adenylate cyclase domain, resulting in the synthesis of cAMP.
authors.affiliation: Division of Structural Biology and Bio-informatics, CSIR-Indian Institute of Chemical Biology, 4 Raja S.C. Mullick Road, Kolkata 700 032, India.
authors.forename: Jayasree
authors.initials: J
authors.lastname: Roy
authors.affiliation: Division of Structural Biology and Bio-informatics, CSIR-Indian Institute of Chemical Biology, 4 Raja S.C. Mullick Road, Kolkata 700 032, India.
authors.forename: Sumit
authors.initials: S
authors.lastname: Sen Santara
authors.affiliation: Division of Structural Biology and Bio-informatics, CSIR-Indian Institute of Chemical Biology, 4 Raja S.C. Mullick Road, Kolkata 700 032, India.
authors.forename: Moumita
authors.initials: M
authors.lastname: Bose
authors.affiliation: Division of Structural Biology and Bio-informatics, CSIR-Indian Institute of Chemical Biology, 4 Raja S.C. Mullick Road, Kolkata 700 032, India.
authors.forename: Supratim
authors.initials: S
authors.lastname: Mukherjee
authors.affiliation: Division of Structural Biology and Bio-informatics, CSIR-Indian Institute of Chemical Biology, 4 Raja S.C. Mullick Road, Kolkata 700 032, India.
authors.forename: Rina
authors.initials: R
authors.lastname: Saha
authors.affiliation: Division of Structural Biology and Bio-informatics, CSIR-Indian Institute of Chemical Biology, 4 Raja S.C. Mullick Road, Kolkata 700 032, India. Electronic address: adaks@iicb.res.in.
authors.forename: Subrata
authors.initials: S
authors.lastname: Adak
mesh_terms: D000262:Adenylyl Cyclases / Q000737:chemistry* / Q000235:genetics; D004795:Enzyme Stability; D005296:Ferrous Compounds / Q000737:chemistry*; D005914:Globins / Q000737:chemistry*; D006418:Heme / Q000737:chemistry*; D006639:Histidine / Q000737:chemistry*; D007700:Kinetics; D018320:Leishmania major / Q000201:enzymology*; D008958:Models, Molecular; D016297:Mutagenesis, Site-Directed
chemical_list: D005296:Ferrous Compounds; D006418:Heme; D006639:Histidine; D005914:Globins; D000262:Adenylyl Cyclases
keywords: Adenylate cyclase; Heme protein; Leishmania; O(2) sensor; Rapid kinetics and mutation; Steady-state catalysis