The ferrous–dioxy complex of Leishmania major globin coupled heme containing adenylate cyclase: The role of proximal histidine on its stability Jayasree Roy, Sumit Sen Santara, Moumita Bose, Supratim Mukherjee, Rina Saha, Subrata Adak ⁎ Division of Structural Biology and Bio-informatics, CSIR-Indian Institute of Chemical Biology, 4 Raja S.C. Mullick Road, Kolkata 700 032, India abstract article info Article history: Received 16 October 2013 Received in revised form 6 January 2014 Accepted 7 January 2014 Available online 12 January 2014 Keywords: Leishmania Heme protein Adenylate cyclase O 2 sensor Steady-state catalysis Rapid kinetics and mutation Recently we have described the globin-coupled heme containing adenylate cyclase from Leishmania major (HemAC-Lm) that shows an O 2 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 O 2 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 576 nm. The distal axial ligand in the deoxygenated ferrous HemAC-Lm is displaced by O 2 at a rate of ~10 s -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 = 6.1 s -1 ). Decomposition of the oxygenated intermediate is independent of O 2 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. © 2014 Elsevier B.V. All rights reserved. 1. Introduction 3′,5′-Cyclic adenosine monophosphate (cAMP) is a ubiquitous second messenger involved in the regulation of several activities in all organisms [1]. cAMP is generated from ATP by a family of enzymes termed as adenylate cyclases (ACs) that can be divided into three classes on the basis of the primary structure of catalytic domains [2]. Class I ACs have been found in enterobacteria. Class II ACs are mainly composed of pathogenic bacterial ACs and are mostly activated by the calmodulin [3,4], whereas Class III ACs are widely distributed from unicellular to multicellular organisms and form the biggest family of ACs. Recently we have described the globin-coupled heme containing adenylate cyclase from Leishmania major (HemAC-Lm) [5]. HemAC-Lm displays spectral properties similar to neuroglobin and cytoglobin. This protein is comprised of a C-terminal catalytic domain containing the ATP binding site, and an N-terminal globin domain that contains binding sites for iron protoporphyrin IX (heme). This protein localizes in the cytosol and oxygen directly stimulates catalytic activity both in vivo and in vitro conditions. O 2 dependent cAMP signaling via protein kinase A plays a key role in cell survival through suppression of oxidative stress under hypoxia. Homology alignment predicts that the heme iron is axially coordinated to the protein via a histidine imidazole, as is the case for all globins, and spectral data show that enzyme is predominantly six-coordinate and low spin in its ferrous state. O 2 appears to bind directly at the distal site of heme iron by displacement of the distal site axial ligand. This O 2 binding may be crucial for changing catalytic AC dimeric structure and inducing catalytic activity. The past few years have witnessed a dramatic increase in the diver- sity of heme-protein sensory domains, including the known heme- containing O 2 -sensors FixL [6], AxPDEA1 [7], NPAS2 [8], EcDOS [9], DosC [10], diguanylate cyclase [11] and HemAT-Bs [12]. Four distinct classes of heme-sensing domains have been discovered e.g. globin, PAS domain, regulatory site of CooA, and guanylate cyclase [12–15]. These domains sequentially control signal transduction domains that include histidine kinases, phosphodiesterases, DNA-binding domains, guanylate cyclases, diguanylate cyclase, and aerotaxis transducers [16–18]. Recently a group of researchers has reported that the heme domain is present in the human soluble adenylate cyclase [19]. As the globin-coupled oxygen sensor protein structures and the signal transduction mechanisms differ from those of other oxygen sensor types, such as FixL and EcDOS containing the heme-bound PAS fold Biochimica et Biophysica Acta 1844 (2014) 615–622 Abbreviations: HemAC-Lm, Leishmania major heme containing adenylate cyclase ⁎ Corresponding author. Tel.: +91 33 2473 6793; fax: +91 33 2473 5197. E-mail address: adaks@iicb.res.in (S. Adak). 1570-9639/$ – see front matter © 2014 Elsevier B.V. All rights reserved. http://dx.doi.org/10.1016/j.bbapap.2014.01.004 Contents lists available at ScienceDirect Biochimica et Biophysica Acta journal homepage: www.elsevier.com/locate/bbapap