Abstract
Bacterial heme nitric oxide/oxygen (H-NOX) domains are nitric oxide (NO) or oxygen sensors. This activity is mediated through binding of the ligand to a heme cofactor. However, H-NOX from Vibrio cholerae (Vc H-NOX) can be easily purified in a heme-free state that is capable of reversibly responding to oxidation, suggesting a heme-independent function as a redox sensor. This occurs by oxidation of Cys residues at a zinc-binding site conserved in a subset of H-NOX homologs. Remarkably, zinc is not lost from the protein upon oxidation, although its ligation environment is significantly altered. Using a combination of computational and experimental approaches, we have characterized localized structural changes that accompany the formation of specific disulfide bonds between Cys residues upon oxidation. Furthermore, the larger-scale structural changes accompanying oxidation appear to mimic those changes observed upon NO binding to the heme-bound form. Thus, Vc H-NOX and its homologs may act as both redox and NO sensors by completely separate mechanisms.
Highlights
The bacterial heme nitric oxide/oxygen (H-NOX) protein family was identified in 2003 through homology to the heme-binding domain of soluble guanylate cyclase [1]
It was previously noted that the three zinc-binding Cys residues identified in S. oneidensis (Cys139, Cys164 and Cys172) were conserved among roughly half of the available hnox sequences from gammaproteobacteria where the fourth ligand (His161) is conserved as either Gln or His [13]
To analyze the degree of conservation of potential zinc-binding sites, a BLASTP search was performed using the Vc H-NOX sequence. 598 sequences were identified with a BLAST score of ≤ 10−20 that fall into four main groups based on a sequence similarity network (SSN) (Figure 1A)
Summary
The bacterial heme nitric oxide/oxygen (H-NOX) protein family was identified in 2003 through homology to the heme-binding domain of soluble guanylate cyclase (sGC) [1]. Bacterial H-NOX proteins bind heme and can be functionally divided into two groups based on the lifestyle of bacteria from which they originate [3]. Those found in strict anaerobes form stable Fe(II)-O2 species. Like sGC, the H-NOX proteins from facultative anaerobes do not stably bind O2 They modulate the activity of histidine kinases (HK) or H-NOX-associated cyclic-di-GMP processing enzymes (HaCE) in a NO-dependent manner, regulating biofilm formation [4,5,6,7], symbiont/host colonization [8] or quorum sensing [9]
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