To survive in hostile environments, organisms activate stress-responsive transcriptional regulators that coordinately increase production of protective factors. Hypoxia changes cellular metabolism and thus activates redox-sensitive as well as oxygen-dependent signal transducers. We demonstrate that Sirtuin 1 (Sirt1), a redox-sensing deacetylase, selectively stimulates activity of the transcription factor hypoxia-inducible factor 2 alpha (HIF-2alpha) during hypoxia. The effect of Sirt1 on HIF-2alpha required direct interaction of the proteins and intact deacetylase activity of Sirt1. Select lysine residues in HIF-2alpha that are acetylated during hypoxia confer repression of Sirt1 augmentation by small-molecule inhibitors. In cultured cells and mice, decreasing or increasing Sirt1 activity or levels affected expression of the HIF-2alpha target gene erythropoietin accordingly. Thus, Sirt1 promotes HIF-2 signaling during hypoxia and likely other environmental stresses.

Neuronal PAS domain protein 2 (NPAS2) is a mammalian transcription factor that binds DNA as an obligate dimeric partner of BMAL1 and is implicated in the regulation of circadian rhythm. Here we show that both PAS domains of NPAS2 bind heme as a prosthetic group and that the heme status controls DNA binding in vitro. NPAS2-BMAL1 heterodimers, existing in either the apo (heme-free) or holo (heme-loaded) state, bound DNA avidly under favorably reducing ratios of the reduced and oxidized forms of nicotinamide adenine dinucleotide phosphate. Low micromolar concentrations of carbon monoxide inhibited the DNA binding activity of holo-NPAS2 but not that of apo-NPAS2. Upon exposure to carbon monoxide, inactive BMAL1 homodimers were formed at the expense of NPAS2-BMAL1 heterodimers. These results indicate that the heterodimerization of NPAS2, and presumably the expression of its target genes, are regulated by a gas through the heme-based sensor described here.

To evaluate the contributions of the Gβ-2 arginine to signal transduction in oxygen-sensing heme-PAS domains, we replaced this residue with alanine in Bradyrhizobium japonicum FixL and examined the results on heme-domain structure, ligand binding, and kinase regulation. In the isolated R220A BjFixL heme-PAS domain, the iron−histidine bond was increased in length by 0.31 Å, the heme flattened even without a ligand, and the interaction of a presumed regulatory loop (the FG loop) with the helix of heme attachment was weakened. Binding of carbon monoxide was similar for ferrous BjFixL and R220A BjFixL. In contrast, the level of binding of oxygen was dramatically lower (K d ∼ 1.5 mM) for R220A BjFixL, and this was manifested as 60- and 3-fold lower on- and off-rate constants, respectively. Binding of cyanide followed the same pattern as binding of oxygen. The catalytic activity was 3−4-fold higher in the “on-state” unliganded forms of R220A BjFixL than in the corresponding BjFixL species. Cyanide regulation of this activity was strongly impaired, but some inhibition was nevertheless preserved. Carbon monoxide and nitric oxide regulation, although weak in BjFixL, were abolished from R220A BjFixL. We conclude that the Gβ-2 arginine assists in the binding of oxygen to BjFixL but does not accomplish this by stabilizing the oxy form. This arginine is not absolutely required for regulation, although it is important for shifting a pre-existing kinase equilibrium toward the inactive state on binding of regulatory ligands. These findings support a regulatory model in which the heme-PAS domain operates as an ensemble that couples to the kinase rather than a mechanism driven by a single central switch.

Phosphorylation of the transcription factor RmFixJ is the key step in the hypoxic induction of Sinorhizobium meliloti nitrogen fixation genes. Oxygen regulates this process by binding reversibly to RmFixL, a heme protein kinase whose deoxy form catalyzes the phosphoryl transfer from ATP to RmFixJ. Here we present the first quantitative measure of the extent by which various heme ligands inhibit the turnover of RmFixJ to phospho-RmFixJ. We also quantitate the inhibition by ligands of the reaction of RmFixL with ATP, in the absence of RmFixJ, to form phospho-RmFixL, i.e., the "autophosphorylation". Phospho-RmFixL formed from autophosphorylation will transfer its phosphoryl group to RmFixJ in an oxygen-independent "phosphotransfer." Here we show that the mode of substrate presentation, i.e., simultaneous versus sequential, influences the regulation of phosphoryl transfer by heme status. Inhibition factors for O(2), CO, NO, CN(-), and imidazole in the presence of RmFixJ are drastically different from the inhibition of autophosphorylation by the same ligands. Oxidation of the heme iron in unliganded RmFixL is known to have no effect on either of the sequential reactions; yet oxidation causes a 100-fold decrease in RmFixJ turnover when ATP and RmFixJ are presented simultaneously. The profound difference between the regulation of isolated RmFixL versus the complex of RmFixL with RmFixJ shows that interaction of a response regulator with its histidine-kinase partner need not be limited to the enzymatic regions of the histidine kinase, but can extend also to its sensory domain.