文摘
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鈥揙 stretching frequency. To investigate the cause of this spectroscopic characteristic, the Fe鈥揅O and C鈥揙 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 FeII d蟺 electrons into the CO 蟺* 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 谓(C鈥揙) of up to 70 cm鈥? relative to the WT protein, with minimal changes in 谓(Fe鈥揅O). Taken together, these results show that the electrostatic interactions in the Tt H-NOX binding pocket are primarily responsible for the high 谓(C鈥揙) by decreasing the Fe d蟺 鈫?CO 蟺* back-donation and suggest that the dominant mechanism by which this family modulates the FeII鈥揅O bond likely involves the YxSxR motif.