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Heme-Nitric Oxide/Oxygen binding (H-NOX) proteins are a family of gas sensing proteins that are prevalent in prokaryotes and higher eukaryotes. H-NOX proteins were initially identified as homologs of the heme domain of the mammalian nitric oxide receptor soluble guanylate cyclase (sGC).


Biological function of H-NOX proteins

Genomic analyses reveal that prokaryotic H-NOX domains are associated with histidine kinases, diguanylate cyclases, and methyl-accepting chemotaxis proteins, suggesting involvement in prokaryotic signaling processes. Uncovering the biological function of these H-NOX domains, particularly in pathogens, is currently an area of intense investigation in our lab.


Ligand affinity and selectivity

H-NOX proteins exhibit remarkable diatomic ligand selectivity: proteins from facultative aerobes exclusively bind NO, like sGC, whereas the H-NOX domains of most obligate anaerobes bind NO and O2. Structural and biochemical studies have shed light on the mechanism of ligand selectivity and highlighted the importance of a hydrogen-bonding network in the distal heme pocket to stabilize O2 as a ligand. Our lab uses a variety of spectroscopic, kinetic, and structural techniques to probe how changes in protein conformation, dynamics, heme distortion, and protein tunnels affect ligand affinity and selectivity.

Molecular Mechanism of H-NOX Signal Transduction

Prokaryotic H-NOX domains are sensors for diatomic gases and as such transfer this sensory input to another signaling partner in the cell. Our lab is investigating the conformational changes that occur when ligands bind to the H-NOX protein and how these are communicated to partner proteins (histidine kinases, diguanylate cyclases, or methyl accepting chemotaxis proteins) to initiate a cascade of signaling events in the cell.

H-NOX Tool Development

The H-NOX protein from Caldanaerobacter subterraneus (formerly Thermoanaerobacter tengcongensis) is a small, readily expressed, remarkably stable protein that can accept a variety of porphyrin analogs in the heme binding pocket. These characteristics inspired our interest in using this porphyrin-binding protein as a biological platform for the development of novel biotechnological tools. One such application was to create an O2-sensor by substituting the natural iron porphyrin with a ruthenium analog, which exhibits O2-dependent phosphorescence. Currently, we are working on utilizing H-NOX proteins to design new tools for medical or biotechnological applications.


Lemon CM and Marletta MA. Corrole-substituted fluorescent heme proteins. Inorg. Chem. 2021

Guo Y and Marletta MA. Structural insight into H-NOX gas sensing and cognate signaling protein regulation. ChemBioChem 2019.

Guo Y, Cooper MM, Bromberg R, Marletta MA. A dual H-NOX signaling system in Saccharophagus degradans. Biochemistry 2018.

Hespen CWBruegger JJ, Guo Y, Marletta MA. Native alanine substitution in the glycine hinge modulates conformational flexibility of Heme Nitric oxide/Oxygen (H-NOX) sensing proteins. ACS Chem. Bio. 2018.
Guo Y, Iavarone AT, Cooper MM, Marletta MA. Mapping the H-NOX/HK Binding Interface in Vibrio cholerae by Hydrogen/Deuterium Exchange Mass Spectrometry. Biochemistry 2018.
Guo Y, Suess DLM, Herzik MA, Iavarone AT, Britt RD, Marletta MA. Regulation of nitric oxide signaling by formation of a distal receptor–ligand complex. Nat Chem Biol. 2017.

Hespen CW, Bruegger JJ, Phillips-Piro CM, Marletta MA. Structural and Functional Evidence Indicates Selective Oxygen Signaling in Caldanaerobacter subterraneus H-NOX. ACS Chem Biol. 2016 Jun 21.

Rao M, Smith BC, Marletta MA. Nitric oxide mediates biofilm formation and symbiosis in Silicibacter sp. Strain TrichCH4B. mBio 2015, 6:e00206-15.


Herzik MA Jr, Jonnalagadda R, Kuriyan J, Marletta MA. Structural insights into the role of iron-histidine bond cleavage in nitric oxide-induced activation of H-NOX gas sensor proteins. Proc Natl Acad Sci USA. 2014, 111, E4156-64.


Nierth A, Marletta MA. Direct meso-alkynylation of metalloporphyrins through gold catalysis for hemoprotein engineering. Angew Chem Int Ed Engl 2014, 53:2611-4.


Plate L, Marletta MA. Phosphorylation-dependent derepression by the response regulator HnoC in the Shewanella oneidensis nitric oxide signaling network. Proc Natl Acad Sci USA 2013, 110:E4648-57.


Weinert EE, Phillips-Piro CM, Marletta MA. Porphyrin π-stacking in a heme protein scaffold tunes gas ligand affinity. J Inorg Biochem 2013, 127:7-12.


Ivanisevic J, Zhu ZJ, Plate L, Tautenhahn R, Chen S, O'Brien PJ, Johnson CH, Marletta MA, Patti GJ, Siuzdak G. Toward 'omic scale metabolite profiling: a dual separation-mass spectrometry approach for coverage of lipid and central carbon metabolism. Anal Chem 2013, 85:6876-84.


Plate L, Marletta MA. Nitric oxide-sensing H-NOX proteins govern bacterial communal behavior. Trends Biochem Sci 2013, 38:566-75. Rev.


Winter MB, Woodward JJ, Marletta MA. An Escherichia coli expression-based approach for porphyrin substitution in heme proteins. Methods Mol Biol 2013, 987:95-106.


Winter MB, Klemm PJ, Phillips-Piro CM, Raymond KN, Marletta MA. Porphyrin-substituted H-NOX proteins as high-relaxivity MRI contrast agents. Inorg Chem 2013, 52:2277-9.


Plate L, Marletta MA. Nitric oxide modulates bacterial biofilm formation through a multicomponent cyclic-di-GMP signaling network. Mol Cell 2012, 46:449-60.


Winter MB, Herzik MA Jr, Kuriyan J, Marletta MA. Tunnels modulate ligand flux in a heme nitric oxide/oxygen binding (H-NOX) domain. Proc Natl Acad Sci USA 2011, 108:E881-9.


Weinert EE, Phillips-Piro CM, Tran R, Mathies RA, Marletta MA. Controlling Conformational Flexibility of an O2-binding N-NOX Domain. Biochemistry 2011, 50:6832-40.


Tran R, Weinert EE, Boon EM, Mathies RA, Marletta MA. Determinants of the Heme-CO Vibrational Modes in the H-NOX Family. Biochemistry 2011, 50:6519-30.


Olea C, Kuriyan J, Marletta MA. Modulating heme redox potential through protein-induced porphyrin distortion. J Am Chem Soc 2010, 132:12794-5.


Carlson HK, Vance RE, Marletta MA. H-NOX regulation of c-di-GMP Metabolism and Biofilm Formation in Legionella pneumophila. Mol Microbiol 2010, 77:930-42.


Wang Y, Dufour YS, Carlson HK, Donohue TJ, Marletta MA, Ruby EG. H-NOX-mediated nitric oxide sensing modulates symbiotic colonization by Vibrio fischeri. Proc Natl Acad Sci USA 2010, 107:8375-80.


Winter MB, McLaurin EJ, Reece SY, Olea C, Nocera DG, Marletta MA. Ru-Porphyrin Protein Scaffolds for Sensing O2. J Am Chem Soc 2010, 132:5582-83.


Olea C Jr, Herzik MA Jr, Kuriyan J, Marletta MA. Structural insights into the molecular mechanism of H-NOX activation.

Protein Sci 2010, 19:881-7.


Weinert EE, Plate L, Whited CA, Olea C Jr, Marletta MA. Determinants of Ligand Affinity and Heme Reactivity in H-NOX Domains. Angew Chem Int Ed Engl 2010, 49:720-23.


Erbil WK, Price MS, Wemmer DE, Marletta MA. A structural basis for H-NOX signaling in Shewanella oneidensis by trapping a histidine kinase inhibitory conformation. Proc Natl Acad Sci USA 2009, 106:19753-60.


Carlson HK, Plate L, Price MS, Allen JJ, Shokat KM, Marletta MA. Use of a semisynthetic epitope to probe histidine kinase activity and regulation. Anal Biochem 2010, 397:139-43.


Tran R, Boon EM, Marletta MA, Mathies RA. Resonance Raman spectra of an O2-binding H-NOX domain reveal heme relaxation upon mutation. Biochemistry 2009, 48:8568-77.


Olea C, Boon EM, Pellicena P, Kuriyan J, Marletta MA. Probing the Function of Heme Distortion in the H-NOX Family. ACS Chem Biol 2008, 3:703-710.


Price MS, Chao LY, Marletta MA. Shewanella oneidensis MR-1 H-NOX Regulation of a Histidine Kinase by Nitric Oxide. Biochemistry 2007, 46:13677-83.


Boon EM, Davis JH, Tran R, Karow DS, Huang SH, Pan D, Miazgowicz MM, Mathies RA, Marletta MA. Nitric oxide binding to prokaryotic homologs of the soluble guanylate cyclase beta1 H-NOX domain. J Biol Chem 2006, 281:21892-902.


Boon EM, Marletta MA. Sensitive and Selective Detection of Nitric Oxide Using an H-NOX Domain. J Am Chem Soc 2006, 128:10022-3.


Boon EM, Marletta MA. Ligand discrimination in soluble guanylate cyclase and the H-NOX family of heme sensor proteins. Curr Opin Chem Biol 2005, 9:441-6.


Boon EM, Huang SH, Marletta MA. A molecular basis for NO selectivity in soluble guanylate cyclase. Nat Chem Biol 2005, 1:53-9.


Boon EM, Marletta MA. Ligand specificity of H-NOX domains: from sGC to bacterial NO sensors. J Inorg Biochem 2005, 99:892-902.


Karow DS, Pan D, Tran R, Pellicena P, Presley A, Mathies RA, Marletta MA. Spectroscopic characterization of the soluble guanylate cyclase-like heme domains from Vibrio cholerae and Thermoanaerobacter tengcongensis. Biochemistry 2004, 43:10203-11.


Pellicena P, Karow DS, Boon EM, Marletta MA, Kuriyan J. Crystal structure of an oxygen-binding heme domain related to soluble guanylate cyclases. Proc Natl Acad Sci USA 2004, 101:12854-9.

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