A “Sliding Scale Rule” for Selectivity among NO, CO, and O₂ by Heme Protein Sensors

Abstract

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.

Gas Sensing and Signaling in the PAS-Heme Domain of the Pseudomonas aeruginosa Aer2 Receptor

“…For example, the histidine kinase FixL is inhibited by O 2 but not by CO or NO, even though it binds these two more tightly (29). Protohemes generally bind O 2 , CO, and NO with relative affinities of 1, 10 3 , and 10 6 , respectively (30,31). In this study, we determined that the O 2 and CO affinities of the isolated Aer2 PAS domain are 16 M and 2 M, respectively (Fig.…”

“…5b). This is not surprising, since heme-CO binding does not require amino acid stabilization due to its high inherent affinity for heme (30). However, heme-CO binding did not predict function.…”

NO• Binds Human Cystathionine β-Synthase Quickly and Tightly

“…In other words, in heme proteins following this rule the ratios for NO ⅐ :CO:O 2 dissociation constants are invariantly 1:ϳ10 3 :ϳ10 6 (36). However, a leveling effect on the ratio between NO ⅐ and CO affinities has been observed for heme proteins with strong field heme ligands, such as cysteine thiolates (36). It is therefore not surprising that human CBS apparently does not follow the sliding scale rule, based on the K d values determined for NO ⅐ and CO (this work and Refs.…”

“…As a general observation, heme proteins with histidine as the proximal ligand and an apolar distal site follow the so-called "sliding scale rule," such that the affinity for NO ⅐ is ϳ10 3 -fold greater than for CO, which in turn binds ϳ10 3 -fold more tightly than O 2 (36). In other words, in heme proteins following this rule the ratios for NO ⅐ :CO:O 2 dissociation constants are invariantly 1:ϳ10 3 :ϳ10 6 (36).…”

Mechanisms of Human Erythrocytic Bioactivation of Nitrite

“…7A). Additionally, the NO donor DEANONOate was added to RBC-CO solution to ensure HbNO could form with NO replacing CO due to hemoglobin's higher affinity for NO compared with CO (47). DEANONOate was also added to deoxygenated RBCs to determine the exact amount of NO released.…”