Gas sensing is crucial for both prokaryotes and eukaryotes and is primarily performed by heme-based sensors, including H-NOX domains. These systems may provide a new, alternative mode for transporting gaseous molecules in higher organisms, but for the development of such systems, a detailed understanding of the ligand-binding properties is required. Here, we focused on ligand migration within the protein matrix: we performed molecular dynamics simulations on three bacterial (Ka, Ns and Cs) H-NOX proteins and studied the kinetics of CO, NO and O2 diffusion. We compared the response of the protein structure to the presence of ligands, diffusion rate constants, tunnel systems and storage pockets. We found that the rate constant for diffusion decreases in the O2 > NO > CO order in all proteins, and in the Ns > Ks > Cs order if single-gas is considered. Competition between gases seems to seriously influence the residential time of ligands spent in the distal pocket. The channel system is profoundly determined by the overall fold, but the sidechain pattern has a significant role in blocking certain channels by hydrophobic interactions between bulky groups, cation–π interactions or hydrogen bonding triads. The majority of storage pockets are determined by local sidechain composition, although certain functional cavities, such as the distal and proximal pockets are found in all systems. A major guideline for the design of gas transport systems is the need to chemically bind the gas molecule to the protein, possibly joining several proteins with several heme groups together.

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The binding of small gas molecules such as NO and CO plays a major role in the signaling routes of the human body. The sole NO-receptor in humans is soluble guanylyl cyclase (sGC) -a histidine-ligated heme protein, which, upon NO binding, activates a downstream signaling cascade. Impairment of NO-signaling is linked, among others, to cardiovascular and inflammatory diseases. In the present work, we use a combination of theoretical tools such as MD simulations, high-level quantum chemical calculations and hybrid QM/MM methods to address various aspects of NO binding and to elucidate the most likely reaction paths and the potential intermediates of the reaction. As a model system, the H-NOX protein from Shewanella oneidensis (So H-NOX) homologous to the NO-binding domain of sGC is used. The signaling route is predicted to involve NO binding to form a six-coordinate intermediate heme-NO complex, followed by relatively facile His decoordination yielding a fivecoordinate adduct with NO on the distal side with possible isomerization to the proximal side through binding of a second NO and release of the first one. MD simulations show that the His sidechain can quite easily rotate outward into solvent, with this motion being accompanied in our simulations by shifts in helix positions that are consistent with this decoordination leading to significant conformational change in the protein.