Spectroscopic Characterization of the Soluble Guanylate Cyclase-like Heme Domains from Vibrio cholerae and Thermoanaerobacter tengcongensis

Abstract

Soluble guanylate cyclase (sGC) is a nitric oxide- (NO-) sensing hemoprotein that has been found in eukaryotes from Drosophila to humans. Prokaryotic proteins with significant homology to the heme domain of sGC have recently been identified through genomic analysis. Characterization of two of these proteins is reported here. The first is a 181 amino acid protein cloned from Vibrio cholerae (VCA0720) that is encoded in a histidine kinase-containing operon. The ferrous unligated form of VCA0720 is 5-coordinate, high-spin. The CO complex is low-spin, 6-coordinate, and the NO complex is high-spin and 5-coordinate. These ligand-binding properties are very similar to those of sGC. The second protein is the N-terminal 188 amino acids of Tar4 (TtTar4H), a predicted methyl-accepting chemotaxis protein (MCP) from the strict anaerobe Thermoanaerobacter tengcongensis. TtTar4H forms a low-spin, 6-coordinate ferrous-oxy complex, the first of this sGC-related family that binds O2. TtTar4H has ligand-binding properties similar to those of the heme-containing O2 sensors such as AxPDEA1. sGC does not bind O2 despite having a porphyrin with a histidyl ligand like the globins. The results reported here, with sequence-related proteins from prokaryotes but in the same family as the sGC heme domain, show that these proteins have evolved to discriminate between ligands such as NO and O2; hence, we term this family H-NOX domains (heme-nitric oxide/oxygen).

Structural insights into the molecular mechanism of H‐NOX activation

“…The H102G Tt H-NOX mutant was expressed and purified as previously described 4,16 and crystallized in several crystal forms. The crystal structure was solved by molecular replacement in two crystal forms (F1 and F2) and refined to 2.0 and 2.1 Å , respectively.…”

“…Expression and purification of Tt H-NOX H102G were carried out as previously described 4,16 with the addition of 10 mM imidazole to all purification buffers and expression media. It has been demonstrated that heme binding in the axial histidine H-NOX mutants can be rescued with exogenous imidazole.…”

“…Recently, heme domains related to sGC were identified in prokaryotes and were termed Heme-Nitric oxide/OXygen binding (H-NOX) domains. 4,5 These heme domains have also been referred to as H-NOBs. 6 Prokaryotic H-NOX proteins fall into two distinct classes.…”

“…The second class consists of H-NOX domains fused to methyl-accepting chemotaxis proteins. 6,4,7 Homology to the sGC heme domain as well as genomic location adjacent to putative signaling proteins suggests that H-NOX domains likely serve as O 2 and NO sensors in prokaryotes. 4,7 Consistent with this hypothesis, the ferrous NO complex of the H-NOX from the facultative aerobe, Shewanella oneidensis (So H-NOX), which belongs to the first class of H-NOX proteins, inhibits autophosphorylation of a histidine kinase encoded in the same operon, whereas the ferrous unliganded protein has no effect on kinase activity.…”

“…6,4,7 Homology to the sGC heme domain as well as genomic location adjacent to putative signaling proteins suggests that H-NOX domains likely serve as O 2 and NO sensors in prokaryotes. 4,7 Consistent with this hypothesis, the ferrous NO complex of the H-NOX from the facultative aerobe, Shewanella oneidensis (So H-NOX), which belongs to the first class of H-NOX proteins, inhibits autophosphorylation of a histidine kinase encoded in the same operon, whereas the ferrous unliganded protein has no effect on kinase activity. 7 Similar to mammalian sGC, So H-NOX is high spin in the ferrous unliganded state, which then forms a transient six-coordinate complex upon binding NO that rapidly converts to a low-spin five-coordinate ferrous nitrosyl complex [ Fig.…”