Bacterial flavohemoglobin: a molecular tool to probe mammalian nitric oxide biology

Abstract

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.

Figure 1

Schematic of the FlavoHb-catalyzed reaction and a kinetic comparison to human iNOS. (A) The heme domain converts NO and O₂ to nitrate (NO₃⁻), with concomitant oxidation of the ferrous to ferric states. The flavin-containing reductase domain uses electrons from NADPH to reduce the ferric back to ferrous heme, thus allowing enzymatic turnover. (B) A kinetic comparison of FlavoHb and iNOS (iNOS was chosen for comparison because it is the most active NOS isoform (i.e., highest V_max), and therefore the most conservative comparison to FlavoHb).

Figure 2

E. coli FlavoHb is a functional NO-consuming enzyme when expressed in mammalian cells. (A) Confocal immunofluorescence reveals that Flag-FlavoHb exhibits diffuse cytosolic localization in transfected HEK293 cells. The white bar indicates 10 μm. (B) Subcellular fractionation demonstrates that Flag-FlavoHb partitions with cytosolic markers (e.g. GAPDH), and is therefore localized predominantly to the cytosol. (C) HEK293 cells were transfected for 24 h with either empty pCDH or pCDH-Flag-FlavoHb, and cytosolic extracts were analyzed for NADPH consumption via spectrophotometry at 340 nm. Reactions were conducted in a phosphate buffer at 37 °C, pH 7.4, and initiated by adding 5 mM spermine-NONOate (indicated by arrow). (D) The presence of FlavoHb shifts the balance of NO oxidation from uncatalyzed auto-oxidation (NO₂^-) to FlavoHb-catalyzed NO dioxygenation (NO₃⁻), as measured by the Griess reaction in the media of HEK293 cells transfected −/+ iNOS and FlavoHb (***, p < 0.001).

Figure 3

FlavoHb can be used to study NO-dependent stress and signaling in mammalian cells. (A) HEK293 cells expressing either empty vector or FlavoHb were treated with DETA-NO for 18 h, then subjected to the ³H-thymidine uptake assay to assess cell proliferation. (B) As in (A), except cells were co-transfected with either empty vector or iNOS for 24 h, then subjected to the ³H-thymidine uptake assay (**, p < 0.01; ***, p < 0.001 by ANOVA; NS, not significant). (C) Colony formation assay with HEK293 cells expressing either empty vector or FlavoHb. Cells were plated into 6 well dishes (500 cells per well) and incubated with DETA-NO at the indicated concentrations for one week. Colonies were then fixed and visualized by methylene blue staining. (D) HEK293 cells expressing either empty vector or FlavoHb were treated with DETA-NO for 18 h, then subjected to immunoblotting as shown. Txnip suppression and VASP phosphorylation are two established NO-dependent signaling pathways, and are blocked by FlavoHb. (E) RAW264.7 macrophages expressing either empty vector or FlavoHb were stimulated with 500 ng/ml LPS for 18 h, and analyzed for Txnip suppression and COX-2 induction by immunoblotting.