title: Determinants of the heme-CO vibrational modes in the H-NOX family.
abstract: The Heme Nitric oxide/OXygen binding (H-NOX) family of proteins have important functions in gaseous ligand signaling in organisms from bacteria to humans, including nitric oxide (NO) sensing in mammals, and provide a model system for probing ligand selectivity in hemoproteins. A unique vibrational feature that is ubiquitous throughout the H-NOX family is the presence of a high C-O stretching frequency. To investigate the cause of this spectroscopic characteristic, the Fe-CO and C-O stretching frequencies were probed in the H-NOX domain from Thermoanaerobacter tengcongensis (Tt H-NOX) using resonance Raman (RR) spectroscopy. Four classes of heme pocket mutants were generated to assess the changes in stretching frequency: (i) the distal H-bonding network, (ii) the proximal histidine ligand, (iii) modulation of the heme conformation via Ile-5 and Pro-115, and (iv) the conserved Tyr-Ser-Arg (YxSxR) motif. These mutations revealed important electrostatic interactions that dampen the back-donation of the Fe(II) d(π) electrons into the CO π* orbitals. The most significant change occurred upon disruption of the H-bonds between the strictly conserved YxSxR motif and the heme propionate groups, producing two dominant CO-bound heme conformations. One conformer was structurally similar to Tt H-NOX WT, whereas the other displayed a decrease in ν(C-O) of up to ∼70 cm(-1) relative to the WT protein, with minimal changes in ν(Fe-CO). Taken together, these results show that the electrostatic interactions in the Tt H-NOX binding pocket are primarily responsible for the high ν(C-O) by decreasing the Fe d(π) → CO π* back-donation and suggest that the dominant mechanism by which this family modulates the Fe(II)-CO bond likely involves the YxSxR motif.
authors.affiliation: Department of Chemistry, University of California , Berkeley, California 94720, United States.
authors.forename: Rosalie
authors.initials: R
authors.lastname: Tran
authors.forename: Emily E
authors.initials: EE
authors.lastname: Weinert
authors.forename: Elizabeth M
authors.initials: EM
authors.lastname: Boon
authors.forename: Richard A
authors.initials: RA
authors.lastname: Mathies
authors.forename: Michael A
authors.initials: MA
authors.lastname: Marletta
mesh_terms: D020816:Amino Acid Motifs; D001426:Bacterial Proteins / Q000737:chemistry* / Q000235:genetics; D002248:Carbon Monoxide / Q000737:chemistry*; D005296:Ferrous Compounds / Q000737:chemistry; D006420:Hemeproteins / Q000737:chemistry* / Q000235:genetics; D006801:Humans; D006860:Hydrogen Bonding; D008024:Ligands; D016297:Mutagenesis, Site-Directed; D009569:Nitric Oxide / Q000737:chemistry* / Q000235:genetics; D010100:Oxygen / Q000737:chemistry*; D011485:Protein Binding / Q000235:genetics; D017434:Protein Structure, Tertiary / Q000235:genetics; D015398:Signal Transduction / Q000235:genetics; D013059:Spectrum Analysis, Raman; D055672:Static Electricity; D046968:Thermoanaerobacter / Q000737:chemistry* / Q000235:genetics; D014732:Vibration
chemical_list: D001426:Bacterial Proteins; D005296:Ferrous Compounds; D006420:Hemeproteins; D008024:Ligands; D009569:Nitric Oxide; D002248:Carbon Monoxide; D010100:Oxygen