title: Probing the chemotaxis periplasmic sensor domains from Geobacter sulfurreducens by combined resonance Raman and molecular dynamic approaches: NO and CO sensing.
abstract: The periplasmic sensor domains encoded by genes gsu0582 and gsu0935 are part of methyl accepting chemotaxis proteins in the bacterium Geobacter sulfurreducens (Gs). The sensor domains of these proteins contain a heme-c prosthetic group and a PAS-like fold as revealed by their crystal structures. Biophysical studies of the two domains showed that nitric oxide (NO) binds to the heme in both the ferric and ferrous forms, whereas carbon monoxide (CO) binds only to the reduced form. In order to address these exogenous molecules as possible physiological ligands, binding studies and resonance Raman (RR) spectroscopic characterization of the respective CO and NO adducts were performed in this work. In the absence of exogenous ligands, typical RR frequencies of five-coordinated (5c) high-spin and six-coordinated (6c) low-spin species were observed in the oxidized form. In the reduced state, only frequencies corresponding to the latter were detected. In both sensors, CO binding yields 6c low-spin adducts by replacing the endogenous distal ligand. The binding of NO by the two proteins causes partial disruption of the proximal Fe-His bond, as revealed by the RR fingerprint features of 5cFe-NO and 6cNO-Fe-His species. The measured CO and NO dissociation constants of ferrous GSU0582 and GSU0935 sensors reveal that both proteins have high and similar affinity toward these molecules (K(d) approximately = 0.04-0.08 microM). On the contrary, in the ferric form, sensor GSU0582 showed a much higher affinity for NO (K(d) approximately = 0.3 microM for GSU0582 versus 17 microM for GSU0935). Molecular dynamics calculations revealed a more open heme pocket in GSU0935, which could account for the different affinities for NO. Taken together, spectroscopic data and MD calculations revealed subtle differences in the binding properties and structural features of formed CO and NO adducts, but also indicated a possibility that a (5c) high-spin/(6c) low-spin redox-linked equilibrium could drive the physiological sensing of Gs cells.
authors.affiliation: Instituto de Tecnologia Química e Biológica, Universidade Nova de Lisboa, 2780-157 Oeiras, Portugal.
authors.forename: Teresa
authors.initials: T
authors.lastname: Catarino
authors.forename: Miguel
authors.initials: M
authors.lastname: Pessanha
authors.forename: Ariel G
authors.initials: AG
authors.lastname: De Candia
authors.forename: Zélia
authors.initials: Z
authors.lastname: Gouveia
authors.forename: Ana P
authors.initials: AP
authors.lastname: Fernandes
authors.forename: P Raj
authors.initials: PR
authors.lastname: Pokkuluri
authors.forename: Daniel
authors.initials: D
authors.lastname: Murgida
authors.forename: Marcelo A
authors.initials: MA
authors.lastname: Marti
authors.forename: Smilja
authors.initials: S
authors.lastname: Todorovic
authors.forename: Carlos A
authors.initials: CA
authors.lastname: Salgueiro
mesh_terms: D002248:Carbon Monoxide / Q000737:chemistry*; D002633:Chemotaxis; D045302:Geobacter / Q000737:chemistry*; D006418:Heme / Q000737:chemistry; D008024:Ligands; D056004:Molecular Dynamics Simulation; D009569:Nitric Oxide / Q000737:chemistry*; D010084:Oxidation-Reduction; D033903:Periplasmic Proteins / Q000737:chemistry*; D011485:Protein Binding; D017434:Protein Structure, Tertiary; D013059:Spectrum Analysis, Raman
chemical_list: D008024:Ligands; D033903:Periplasmic Proteins; D009569:Nitric Oxide; D006418:Heme; D002248:Carbon Monoxide