Bacterial Heme-Nitric Oxide/Oxygen binding (H-NOX) proteins utilize heme to act as diatomic gas sensors. These proteins often interact with a signaling partner to govern communal behaviors such as quorum sensing and biofilm formation in response to nitric oxide in facultative anaerobes. H-NOX from Vibrio cholerae (Vc H-NOX) in the Fe(III) and Fe(II)-NO states was demonstrated to inhibit the activity of a cognate histidine kinase (HK). Heme-free Vc H-NOX could also act as an inhibitor of HK signaling through reversible oxidation of cysteine residues at a zinc binding site, suggesting that H-NOX proteins may act as redox sensors in some organisms. The zinc-binding Cys residues are conserved across numerous species, including the aquatic organism and obligate aerobe, Caulobacter crescentus (Cc H-NOX). Using UV-Vis spectroscopy, our lab has characterized heme complexes from Cc H-NOX as purified, reduced, CO-bound, and NO-bound. ICP-OES has provided insight to the degree of heme binding in Cc H-NOX samples as well as zinc content. Circular dichroism has provided an estimate of secondary structure and a melt curve using single wavelength readings with variable temperatures. Lastly, we have generated a knockout mutant to investigate the importance of H-NOX in biofilm regulation and the impact this knockout has on the Caulobacter crescentus proteome. Our results have shown complete zinc binding for Cc H-NOX, the formation of a stable NO-complex, and a mild hyper-biofilm phenotype among Caulobacter crescentus mutants. The mutant also exhibits differences in protein expression levels and phosphorylation reflecting signaling pathways altered in the absence of H-NOX. This has led to an improved understanding of the roles of H-NOX signaling across bacterial species.
This work is supported by an award from the National Science Foundation award number 2103676.
