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Nitric oxide (NO) plays a prominent role in mammals as a cytotoxic agent released by macrophages and as a signaling molecule involved in neurotransmission, blood flow, and platelet aggregation. NO signaling in vivo begins with its synthesis by nitric oxide synthases (NOS). Mammalian NOS enzymes are comprised of an oxygenase domain containing heme and tetrahydrobiopterin (H4B) cofactors, a reductase domain that binds flavin mononucleotide (FMN) and flavin adenine dinucleotide (FAD), and an intervening calmodulin binding region. NOS enzymes catalyze the oxidation of arginine to NO and citrulline using oxygen and NADPH as cosubstrates in a two-step reaction with N-hydroxyarginine (NHA) as an intermediate. Both steps occur in the heme-containing oxidase domain of NOS, which is fed electrons from the flavin-containing reductase domain. However, rate limiting electron transfer masks observation of the activated oxygen intermediates responsible for arginine and NHA oxidation. Knowledge of these intermediates is crucial to understanding the catalytic mechanism of NOS enzymes. We are developing novel spectroscopic techniques to rapidly reduce the heme and directly observe these intermediates. We have also generated unnatural substrate and heme analogs and developed methods for incorporation of unnatural amino acids, H4B, and heme analogs. This approach allows the unique ability to probe the structure-function and thermodynamic-kinetic relationships of oxygen activation in NOS enzymes.



Genome sequencing has revealed that many bacterial species also possess NOS-like proteins. While most of these bacterial NOS proteins contain only a single domain with high sequence homology to the oxygenase domain of mammalian NOS, a handful of recently-discovered bacterial NOS proteins are fused to reductase domains. We are generally interested in these novel bacterial NOS proteins. Do these bacterial NOS proteins produce NO? What substrates and cofactors are required for their activity? How do the mechanisms of NO synthesis compare and contrast to the mammalian NOS enzymes? What role do these bacterial NOS proteins play in bacterial physiology?

Publications (since 2002):


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


Campbell MG, Smith BC, Potter CS, Carragher B, Marletta MA. Molecular architecture of mammalian nitric oxide synthases. Proc Natl Acad Sci USA. 2014, 111:E3614-23.


Smith BC, Underbakke ES, Kulp DW, Schief WR, Marletta MA. Nitric oxide synthase domain interfaces regulate electron transfer and calmodulin activation. Proc Natl Acad Sci USA 2013, 110:E3577-86.


Smith BC, Fernhoff, NB, Marletta MA. Mechanisms and kinetics of nitric oxide synthase auto-S-nitrosation and inactivation. Biochemistry 2012, 51:1028-40.


Stoll S, Nejatyjahromy Y, Woodward JJ, Ozarowski MA, Britt RD. Nitric oxide synthase stabilizes the tetrahydropbiopterin cofactor radical by controlling its protonation state. J Am Chem Soc 2010, 132:11812-23.


Woodward JJ, Nejatyjahromy Y, Britt RD, Marletta MA. Pterin-centered radical as a mechanistic probe of the second step of nitric oxide synthase. J Am Chem Soc 2010, 132:5105-13.


Agapie T, Suseno S, Woodward JJ, Stoll S, Britt RD, Marletta MA. NO formation by a catalytically self-sufficient bacterial nitric oxide synthase from Sorangium cellulosum. Proc Natl Acad Sci USA 2009, 106:16221-6.


Reece SY, Woodward JJ, Marletta MA. Synthesis of nitric oxide by the NOS-like protein from Deinococcus radiodurans: A direct role for tetrahydrofolate. Biochemistry 2009, 48:5483-91.


Martin NI, Woodward JJ, Winter MB, Marletta MA. 4,4-Difluorinated analogues of l-arginine and N(G)-hydroxy-l-arginine as mechanistic probes for nitric oxide synthase. Bioorg Med Chem Lett 2009, 19:1758-62.


Woodward JJ, Chang MM, Martin NI, Marletta MA.  The second step of the nitric oxide synthase reaction: evidence for ferric-peroxo as the active oxidant. J Am Chem Soc 2009, 131:297-305.


Martin NI, Beeson WT, Woodward JJ, Marletta MA. N(G)-Aminoguanidines from primary amines and the preparation of nitric oxide synthase inhibitors. J Med Chem 2008, 51:924-31.


Martin NI, Woodward JJ, Winter MB, Beeson WT, Marletta MA. Design and synthesis of C5 methylated L-arginine analogues as active site probes for nitric oxide synthase. J Am Chem Soc 2007, 129:12563-70.


Woodward JJ, Martin NI, Marletta MA. An Escherichia coli expression-based method for heme substitution. Nat Methods 2007, 4:43-5.


Martin NI, Woodward JJ, Marletta MA. NG-hydroxyguanidines from primary amines. Org Lett 2006, 8:4035-8.


Erwin PA, Mitchell DA, Sartoretto J, Marletta MA, Michel T. Subcellular targeting and differential S-nitrosylation of endothelial nitric-oxide synthase. J Biol Chem 2006 281:151-7.


Mitchell DA, Erwin PA, Michel T, Marletta MA. S-Nitrosation and regulation of inducible nitric oxide synthase. Biochemistry 2005, 44:4636-47.


Luzzi SD, Marletta MA. L-arginine analogs as alternate substrates for nitric oxide synthase. Bioorg Med Chem Lett 2005, 15:3934-41.


Udit AK, Belliston-Bittner W, Glazer EC, Nguyen YH, Gillan JM, Hill MG, Marletta MA, Goodin DB, Gray HB. Redox couples of inducible nitric oxide synthase. J Am Chem Soc 2005, 127:11212-3.


Gribovskaja I, Brownlow KC, Dennis SJ, Rosko AJ, Marletta MA, Stevens-Truss R. Calcium-binding sites of calmodulin and electron transfer by inducible nitric oxide synthase. Biochemistry 2005, 44:7593-601.


Hurshman AR, Krebs C, Edmondson DE, Marletta MA. Ability of tetrahydrobiopterin analogues to support catalysis by inducible nitric oxide synthase: formation of a pterin radical is required for enzyme activity. Biochemistry 2003, 42:13287-303.


Hurshman, AR, Marletta, MA. Reactions catalyzed by the heme domain of inducible nitric oxide synthase: evidence for the involvement of tetrahydrobiopterin in electron transfer. Biochemistry 2002, 41:3439-56.

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