Nitric oxide (NO) is extremely toxic to Clostridium botulinum, but its molecular targets are unknown. Here, we identify a heme protein sensor (SONO) that displays femtomolar affinity for NO. The crystal structure of the SONO heme domain reveals a previously undescribed fold and a strategically placed tyrosine residue that modulates heme-nitrosyl coordination. Furthermore, the domain architecture of a SONO ortholog cloned from Chlamydomonas reinhardtii indicates that NO signaling through cyclic guanosine monophosphate arose before the origin of multicellular eukaryotes. Our findings have broad implications for understanding bacterial responses to NO, as well as for the activation of mammalian NO-sensitive guanylyl cyclase.

It has been previously shown that besides synthesizing nitric oxide (NO),

The coupling between the peroxidase and cyclooxygenase activities of prostaglandin H synthase (PGHS) has been proposed to be mediated by a critical tyrosyl radical through a branched chain mechanism (Dietz, R., Nastainczyk, W., and Ruf, H. H. (1988) Eur. J. Biochem. 171, 321-328). In this study, we have examined the ability of PGHS isoform-1 (PGHS-1) tyrosyl radicals to react with arachidonate. Anaerobic addition of arachidonate following formation of the peroxide-induced wide doublet or wide singlet tyrosyl radical led to disappearance of the tyrosyl radicals and emergence of a new EPR signal, which is distinct from known PGHS-1 tyrosyl radicals. The new radical was clearly derived from arachidonate because its EPR line shape changed when 5,6,8,9,11,12,14,15-octadeuterated arachidonate was used. Subsequent addition of oxygen to samples containing the fatty acyl radical resulted in regeneration of tyrosyl radical EPR. In contrast, the peroxide-generated tyrosyl radical in indomethacin-treated PGHS-1 (a narrow singlet) failed to react with arachidonate, consistent with the cyclooxygenase inhibition by indomethacin. These results indicate that the peroxide-generated wide doublet and wide singlet tyrosyl radicals serve as immediate oxidants of arachidonate bound at the cyclooxygenase active site to form a carbon-centered fatty acyl radical, which reacts with oxygen to form a hydroperoxide. These observations represent the first direct evidence of chemical coupling between the peroxidase reaction and arachidonate oxygenation in PGHS-1 and support the proposed role for a tyrosyl radical in cyclooxygenase catalysis.

Selectivity between NO, CO, and O2 is crucial for the physiological function of most heme proteins. Although there is a million-fold variation in equilibrium dissociation constants (KDs), the ratios for NO:CO:O2 binding stay roughly the same, 1:~103:~106 when the proximal ligand is a histidine and the distal site is apolar. For these proteins, there is a “sliding scale rule” for plots of log KD versus ligand type that allows predictions of KD values if one or two are missing. The predicted KD for O2 binding to Ns H-NOX coincides with the value determined experimentally at high pressures. Active site hydrogen bond donors break the rule and selectively increase O2 affinity with little effect on CO and NO binding. Strong field proximal ligands such as thiolate, tyrosinate and imidazolate exert a “leveling” effect on ligand binding affinity. The reported picomolar KD for NO binding to sGC deviates even more dramatically from the sliding scale rule, showing a NO:CO KD ratio of 1: ~108. This deviation is explained by a complex, multi-step process, in which an initial low affinity hexacoordinate NO complex with a measured KD ≈ 54 nM, matching that predicted from the sliding scale rule, is formed initially and then converts to a high affinity pentacoordinate complex. This multi-step 6c to 5c mechanism appears common to all NO sensors that exclude O2 binding in order to capture lower level of cellular NO and prevent its consumption by dioxygenation.