title: Redox-dependent Ligand Switching in a Sensory Heme-binding GAF Domain of the Cyanobacterium Nostoc sp. PCC7120.
abstract: The genome of the cyanobacterium Nostoc sp. PCC7120 carries three genes (all4978, all7016, and alr7522) encoding putative heme-binding GAF (cGMP-specific phosphodiesterases, adenylyl cyclases, and FhlA) proteins that were annotated as transcriptional regulators. They are composed of an N-terminal cofactor domain and a C-terminal helix-turn-helix motif. All4978 showed the highest affinity for protoheme binding. The heme binding capability of All7016 was moderate, and Alr7522 did not bind heme at all. The "as isolated" form of All4978, identified by Soret band (λmax = 427 nm), was assigned by electronic absorption, EPR, and resonance Raman spectroscopy as a hexa-coordinated low spin Fe(III) heme with a distal cysteine ligand (absorption of δ-band around 360 nm). The protoheme cofactor is noncovalently incorporated. Reduction of the heme could be accomplished by chemically using sodium dithionite and electrospectrochemically; this latter method yielded remarkably low midpoint potentials of -445 and -453 mV (following Soret and α-band absorption changes, respectively). The reduced form of the heme (Fe(II) state) binds both NO and CO. Cysteine coordination of the as isolated Fe(III) protein is unambiguous, but interestingly, the reduced heme instead displays spectral features indicative of histidine coordination. Cys-His ligand switches have been reported as putative signaling mechanisms in other heme-binding proteins; however, these novel cyanobacterial proteins are the first where such a ligand-switch mechanism has been observed in a GAF domain. DNA binding of the helix-turn-helix domain was investigated using a DNA sequence motif from its own promoter region. Formation of a protein-DNA complex preferentially formed in ferric state of the protein.
authors.affiliation: From the State Key Laboratory of Agricultural Microbiology, Huazhong Agricultural University, Wuhan 430070, China, the Max Planck Institute for Chemical Energy Conversion, Stiftstrasse 34-36, D-45470 Mülheim, Germany.
authors.forename: Kun
authors.initials: K
authors.lastname: Tang
authors.affiliation: the Max Planck Institute for Chemical Energy Conversion, Stiftstrasse 34-36, D-45470 Mülheim, Germany, Resolv, Faculty for Chemistry and Biochemistry, Ruhr University Bochum, D-44780 Bochum, Germany, and.
authors.forename: Markus
authors.initials: M
authors.lastname: Knipp
authors.affiliation: From the State Key Laboratory of Agricultural Microbiology, Huazhong Agricultural University, Wuhan 430070, China.
authors.forename: Bing-Bing
authors.initials: BB
authors.lastname: Liu
authors.affiliation: the Max Planck Institute for Chemical Energy Conversion, Stiftstrasse 34-36, D-45470 Mülheim, Germany.
authors.forename: Nicholas
authors.initials: N
authors.lastname: Cox
authors.affiliation: the Max Planck Institute for Chemical Energy Conversion, Stiftstrasse 34-36, D-45470 Mülheim, Germany.
authors.forename: Robert
authors.initials: R
authors.lastname: Stabel
authors.affiliation: From the State Key Laboratory of Agricultural Microbiology, Huazhong Agricultural University, Wuhan 430070, China.
authors.forename: Qi
authors.initials: Q
authors.lastname: He
authors.affiliation: From the State Key Laboratory of Agricultural Microbiology, Huazhong Agricultural University, Wuhan 430070, China.
authors.forename: Ming
authors.initials: M
authors.lastname: Zhou
authors.affiliation: the Department of Biologie I, Ludwig-Maximilians-Universität, Menzinger Strasse 67, D-80638 München, Germany.
authors.forename: Hugo
authors.initials: H
authors.lastname: Scheer
authors.affiliation: From the State Key Laboratory of Agricultural Microbiology, Huazhong Agricultural University, Wuhan 430070, China, khzhao@163.com.
authors.forename: Kai-Hong
authors.initials: KH
authors.lastname: Zhao
authors.affiliation: the Max Planck Institute for Chemical Energy Conversion, Stiftstrasse 34-36, D-45470 Mülheim, Germany, wolfgang.gaertner@cec.mpg.de.
authors.forename: Wolfgang
authors.initials: W
authors.lastname: Gärtner
mesh_terms: D000595:Amino Acid Sequence; D001426:Bacterial Proteins / Q000737:chemistry* / Q000502:physiology; D001483:Base Sequence; D000458:Cyanobacteria / Q000378:metabolism*; D004269:DNA, Bacterial / Q000737:chemistry / Q000235:genetics; D006418:Heme / Q000737:chemistry; D006420:Hemeproteins / Q000737:chemistry* / Q000502:physiology; D008024:Ligands; D008969:Molecular Sequence Data; D010084:Oxidation-Reduction; D011401:Promoter Regions, Genetic; D011485:Protein Binding; D017433:Protein Structure, Secondary
chemical_list: D001426:Bacterial Proteins; D004269:DNA, Bacterial; D006420:Hemeproteins; D008024:Ligands; D006418:Heme
keywords: GAF domain; Raman spectroscopy; cyanobacteria; electron paramagnetic resonance (EPR); heme; ligand-binding protein; protein conformation; protein folding; signaling