Gaseous ligand selectivity of the H-NOX sensor protein from Shewanella oneidensis and comparison to those of other bacterial H-NOXs and soluble guanylyl cyclase

Abstract

To delineate the commonalities and differences in gaseous ligand discrimination among the heme-based sensors with Heme Nitric oxide/OXygen binding protein (H-NOX) scaffold, the binding kinetic parameters for gaseous ligands NO, CO, and O2, including KD, kon, and koff, of Shewanella oneidensis H-NOX (So H-NOX) were characterized in detail in this study and compared to those of previously characterized H-NOXs from Clostridium botulinum (Cb H-NOX), Nostoc sp. (Ns H-NOX), Thermoanaerobacter tengcongensis (Tt H-NOX), Vibrio cholera (Vc H-NOX), and human soluble guanylyl cyclase (sGC), an H-NOX analogue. The KD(NO) and KD(CO) of each bacterial H-NOX or sGC follow the “sliding scale rule”; the affinities of the bacterial H-NOXs for NO and CO vary in a small range but stronger than those of sGC by at least two orders of magnitude. On the other hand, each bacterial H-NOX exhibits different characters in the stability of its 6c NO complex, reactivity with secondary NO, stability of oxyferrous heme and autoxidation to ferric heme. A facile access channel for gaseous ligands is also identified, implying that ligand access has only minimal effect on gaseous ligand selectivity of H-NOXs or sGC. This comparative study of the binding parameters of the bacterial H-NOXs and sGC provides a basis to guide future new structural and functional studies of each specific heme sensor with the H-NOX protein fold.

Structural Insight into H‐NOX Gas Sensing and Cognate Signaling Protein Regulation

“…This 6‐coordinate complex then converts to a 5‐coordinate ferrous nitrosyl complex as the strong‐field NO ligand induces the cleavage of the Fe II ‐His bond . It has been observed that the rate of conversion of the 6‐coordinate intermediate to the final 5‐coordinate complex is dependent on the concentration of NO (Figure A, k 2 ). The NO dependence of this transformation was rationalized by invoking the formation of a dinitrosyl intermediate, which could form either a proximal or distal 5‐coordinate complex by dissociation of the opposite NO molecule.…”

Gas Sensing by Bacterial H-NOX Proteins: An MD Study

“…VAL77, ILE78, SER81, TYR82, VAL107 and TYR111 see Figure 4 ) leading to one side of the heme group. For this system, the direct Tunnel 2 , previously suggested to be universal for all H-NOXs, was not observed [ 9 ]. Closer inspection showed that this tunnel is blocked at both ends in Ka .…”

“…Both of these routes are short and provide easy access to both the distal and the proximal sides of the heme. These tunnels may play a major role in the reactivity of sGC [ 36 ], Shewanella oneidensis ( So ) H-NOX [ 9 ] and Vibrio cholera ( Vc ) H-NOX for which ligand-binding to the proximal side has also been suggested. Tunnel 1 , the long apolar tunnel has been visited by ligands at its entrance towards the solvent phase; however, the exit to the distal pocket remained fully blocked by a hydrogen bonding triad (TYR140 with sidechains of TRP9 and ASN74).…”

“…Selective binders can be used for trafficking gaseous ligands: H-NOX proteins (named Heme-Nitric oxide and OXygen binding domain) are one of the six major groups of heme sensor proteins whose bindings with the gaseous messengers cause changes in the downstream effector proteins, evoking various responses [ 9 ]. H-NOXs were already shown to be able to deliver O 2 to hypoxic regions of solid tumors [ 10 ] and accumulate as a multimeric pattern in multiple intracranial glioblastoma models, thus improving the efficacy of radiation therapy [ 10 , 11 ].…”

“…On the experimental side, xenon (Xe) saturation experiments in conjunction with X-Ray diffraction studies found bifurcated Y-shaped tunnels in Ns H-NOX, but no continuous pathway connecting the heme site and the solvent in Cs H-NOX showing that the fold of the protein is not the only determining factor of the ligand migration mechanism [ 20 ]. In contrast, based on the shape of the molecular surfaces, Wu et al proposed that the major and most significant route for ligand entry to the heme group in all H-NOXs is the wide gateway in the plane of the heme group on the side of its propionates [ 9 ]; however, no such route was identified in the case of Cs H-NOX by molecular dynamics simulations [ 21 ]. These studies demonstrated that ligand access does not necessarily require additional channel systems to connect the heme site and the solvent, and that the different protein matrices of H-NOXs, in spite of the identical fold, possess different cavity structures, in which differing sidechain compositions lead to the formation of pockets of distinct positions, sizes and polarities, which could be important for their divergent functions.…”