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Bacterial flavohemoglobin: a molecular tool to probe mammalian nitric oxide biology
Michael T. Forrester*1,3, Christine E. Eyler2,3, 4, and Jeremy N. Rich4
1Department of Biochemistry, Duke University Medical Center, Durham, NC, USA
2Department of Pharmacology and Cancer Biology, Duke University Medical Center, Durham, NC, USA
3Medical Scientist Training Program, Duke University Medical Center, Durham, NC, USA
4Department of Stem Cell Biology and Regenerative Medicine, Lerner Research Institute, Cleveland Clinic, Cleveland, OH, USA
BioTechniques, Vol. 50, No. 1, January 2011, pp. 41–45
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Supplementary Material

A wide range of mammalian signaling and stress pathways are mediated by nitric oxide (NO), which is synthesized in vivo by the nitric oxide synthase (NOS) family of enzymes. Experimental manipulations of NO are frequently achieved by either inhibition or activation of endogenous NOS or via providing exogenous NO sources. On the contrary, many microbes consume NO via flavohemoglobin (FlavoHb), a highly efficient NO-dioxygenase that protects from nitrosative stress. Here we report a novel resource for studying NO in mammalian cells by heterologously expressing Escherichia coli FlavoHb within a lentiviral delivery system. This technique boosts endogenous cellular consumption of NO, thus providing a simple and efficacious approach to studying mammalian NO biology that can be employed as both a primary experimental and confirmatory tool.

The diatomic gas nitric oxide (NO) plays a multitude of roles in mammalian, plant, and microbial biology (1-4). In models of infection, mammalian-derived NO facilitates microbial killing (5,6), indicating that NO is a central element of innate immunity. In return, bacteria and fungi possess two major systems to metabolize NO: the flavorubredoxin NO-reductase (7) and flavohemoglobin (FlavoHb) NO-dioxygenase (8-10) enzymes, which operate under strictly anaerobic and aerobic/microaerophilic conditions, respectively. The importance of NO metabolism in the prokaryotic life cycle is evidenced by the evolution of the FlavoHb family nearly 2 billion years ago (11), suggesting that microbes have been coping with nitrosative stress long before mammals existed.

A wealth of studies over the past decade has elucidated the mechanism and function of FlavoHb in bacteria and fungi. Under aerobic/microaerophilic conditions, FlavoHb converts NO and O2 into nontoxic nitrate (NO3) with concomitant oxidation of ferrous (Fe2+) to ferric (Fe3+) heme within FlavoHb (9). The active site flavin adenine dinucleotide (FAD) supports one-electron reduction back o the ferrous state, driven by electrons from NADH or NADPH (Figure 1A). The enzyme is not known to react with substrates other than NO. Further, FlavoHb is transcriptionally induced by NO in many bacterial and fungal pathogens, and has been shown to play a central role in protection from NO in multiple pathogenic microbes (5,10)(12-19).

In contrast to microbes, the FlavoHb gene is not present in metazoans or mammals. On the contrary, mammals synthesize NO via three conserved nitric oxide synthase (NOS) isoforms: iNOS/NOS2 (“inducible”), eNOS/NOS3 (“endothelial”), and nNOS/NOS1 (“neuronal”), each of which plays distinct biological roles (20). Though the overall importance of NO is widely appreciated, techniques to determine the cellular roles of NO have relied predominantly on manipulating NOS expression or activity, most frequently via arginine-based NOS inhibitors. While this approach is undoubtedly powerful, there are several drawbacks to NOS inhibitors: (i) they rarely exhibit strong isoform selectivity, with the exception of some iNOS inhibitors such as 1400W (21) and BYK191023 (22), (ii) they are typically arginine analogs, and several studies have suggested they may perturb arginine uptake or metabolism (23-25),and (iii) NOS-independent sources of NO are unaffected. On the contrary, NOS over expressionor administration of NO-donor compounds may result in supraphysiologic levels of NO. Reliance on such methods might therefore lead to aberrant cellular effects, thus confounding the interpretation of NO's roles in mammalian cells. A technique to selectively deplete NO—independently of NO source—would therefore be of significant utility in studies of NO biology.

Given the remarkable specificity and catalytic efficiency of bacterial NO-consuming enzymes, we hypothesized that heterologous expression of such an enzyme in mammalian cells might be a useful tool to interrogate the biological roles of NO. While the flavorubredoxin is a potent NO reductase, it requires exclusively anaerobic conditions. On the contrary, FlavoHb operates under a wide range of O2 concentrations, suggesting it might also operate in mammalian cells. Furthermore, flavorubredoxin consists of two separate polypeptides (the NO reductase and flavorubredoxin reductase), whereas FlavoHb is a 44-kD protein encoded by a single gene, thus simplifying the strategy of heterologous expression. As compared in Figure 1B, the kinetic parameters of Escherichia coli FlavoHb (26) are far superior to even the most robust mammalian NOS isoform, iNOS (27). We therefore chose to focus our efforts on FlavoHb.

Materials and methods


All materials were from Sigma-Aldrich (St. Louis, MO, USA) unless otherwise indicated. LPS was from E. coli strain 026:B6. Protease inhibitor (PI) cocktail was from Roche (Indianapolis, IN, USA). “NONOate” NO donors and 1400W were from Cayman Chemical and freshly prepared in 10 mM NaOH. Sources of antibodies were: α-Txnip/Vdup1 mouse MAb (Cat. no. K0205–3; MBL International, Woburn, MA, USA), α-GAPDH mouse MAb (Cat. no. MAB374; Millipore, Billerica, MA, USA), α-COX2 rabbit polyclonal antibody (pAb) (Cat. no. 4842; Cell Signaling Technology, Boston, MA, USA), α-VASP rabbit pAb (Cat. no. 3112; Cell Signaling Technology), α-phospho-VASP rabbit pAb (Cat. no. 3111; Cell Signaling Technology), α-iNOS mouse MAb (Cat. no. 610328; BD Biosciences, San Jose, CA, USA), α-Flag M2 mouse MAb for immunoblotting (Cat. no. F1804; Sigma-Aldrich), α-Flag rabbit pAb for immunofluorescence (Cat. no. 2368; Cell Signaling), α-catalase mouse MAb (Cat. no. C0979; Sigma-Aldrich), α-PCNA mouse MAb (Cat. no. 2586; Cell Signaling).

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