Dissection of Nitric Oxide binding pathway in Nitrobindins reveals hidden structural details

Nitric Oxide (NO) rebinding after a very short laser pulse (<10−12 s) has been investigated in nitrobindins (Nbs) from plant Arabidopsis thaliana (At[sbnd]Nb) and zebrafish Danio rerio (Dr-Nb). Nbs are ten-stranded anti-parallel all-β-barrel heme-proteins present along the evolutionary ladder, from bacteria to man. They display an open distal heme pocket, easily accessible to the bulk solvent, since the histidine residue, facing the distal side of the heme in most all-α-helical hemoproteins, is absent. Further, they display a proximal histidine residue as the fifth coordinating ligand to the heme-Fe atom, which is severely weakened, or even cleaved, upon NO binding; in particular, spectroscopic evidence shows that in NO-bound Dr-Nb(II) the proximal bond is absent, whereas in At-Nb(II) NO binding induces a structural stress. Noteworthy, NO geminate recombination is very fast in both Nb(II) proteins, being essentially completed within 3 × 10−11 s and involving >80 % of the photo-dissociated molecules. This behavior is drastically different from what observed in α-helical heme-proteins, such as myoglobin and hemoglobin, which show a multiphasic and slower geminate rebinding process. Thus, it suggests that the open heme pocket does not facilitate the outward diffusion of the photo-dissociated NO, but instead the lack of residues in the immediate proximity likely helps the ligand to keep an optimal space configuration for fast rebinding. The dissection of outward and inward ligand pathway steps allows a better comprehension of determinants for NO binding to heme-proteins, a crucial event for blood flow regulation in poorly vascularized tissues, like the eye's retina.