Role of conserved Fα-helix residues in the native fold and stability of the kinase-inhibited oxy state of the oxygen-sensing FixL protein from Sinorhizobium meliloti

Reynolds, Mark F.; Ackley, Lindsey; Blizman, Alice; Lutz, Zachary; Manoff, David; Miles, Matthew B.; Pace, Matthew; Patterson, Joseph R.; Pozzessere, Nicholas; Saia, Kathryn; Sato, Rei; Smith, Danielle M.; Tarves, Paul C.; Weaver, Matthew; Sieg, Kristina; Lukat-Rodgers, Gudrun S.; Rodgers, Kenton R.

doi:10.1016/j.abb.2009.02.011

Cited by 6 publications

(14 citation statements)

References 58 publications

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“…Fig 4, trace A is the rR spectrum of FVs suspended in phosphate buffer saline (PBS) with no nitrate or nitrite added. These rR bands are typical of a five-coordinate ferric heme (Reynolds et al, 2009). The oxidation-state (or π* electron density) marker band (ν 4 ) was observed at 1372 cm −1 , consistent with ferric heme.…”

Section: Resultsmentioning

confidence: 99%

Plasmodium falciparum: Nitric oxide modulates heme speciation in isolated food vacuoles

Ostera¹,

Tokumasu²,

Teixeira³

et al. 2011

Experimental Parasitology

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Nitric oxide (NO) and NO-derived reactive nitrogen species (RNS) are present in the food vacuole (FV) of P. falciparum trophozoites. The product of PFL1555w, a putative cytochrome b5, localizes in the FV membrane, similar to what was previously observed for the product of PF13_0353, a putative cytochrome b5 reductase. These two gene products may contribute to NO generation by denitrification chemistry from nitrate and/or nitrite present in the erythrocyte cytosol. The possible coordination of NO to heme species present in the food vacuole was probed by resonance Raman spectroscopy. The spectroscopic data revealed that in situ generated NO interacts with heme inside the intact FVs to form ferrous heme nitrosyl complexes that influence intra-vacuolar heme solubility. The formation of heme nitrosyl complexes within the FV is a previously unrecognized factor that could affect the equilibrium between soluble and crystallized heme within the FV in vivo.

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Section: Resultsmentioning

confidence: 99%

Plasmodium falciparum: Nitric oxide modulates heme speciation in isolated food vacuoles

Ostera¹,

Tokumasu²,

Teixeira³

et al. 2011

Experimental Parasitology

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“…B) The heme active site of Bj FixL. Hydrogen bond interactions to the heme propionate groups have a significant role in shifting the FG loop conformation for oxy-FixL, slowing autophosphorylation [124, 133-135, 150-152]. C) Protein constructs of Bj FixL, Re FixL and Sm FixL.…”

Section: Highlightsmentioning

confidence: 99%

Oxygen sensing strategies in mammals and bacteria

Taabazuing

Knapp

2014

Journal of Inorganic Biochemistry

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The ability to sense and adapt to changes in pO2 is crucial for basic metabolism in most organisms, leading to elaborate pathways for sensing hypoxia (low pO2). This review focuses on the mechanisms utilized by mammals and bacteria to sense hypoxia. While responses to acute hypoxia in mammalian tissues lead to altered vascular tension, the molecular mechanism of signal transduction is not well understood. In contrast, chronic hypoxia evokes cellular responses that lead to transcriptional changes mediated by the hypoxia inducible factor (HIF), which is directly controlled by post-translational hydroxylation of HIF by the non-heme Fe(II)/αKG-dependent enzymes FIH and PHD2. Research on PHD2 and FIH is focused on developing inhibitors and understanding the links between HIF binding and the O2 reaction in these enzymes. Sulfur speciation is a putative mechanism for acute O2-sensing, with special focus on the role of H2S. This sulfur-centered model is discussed, as are some of the directions for further refinement of this model. In contrast to mammals, bacterial O2-sensing relies on protein cofactors that either bind O2 or oxidatively decompose. The sensing modality for bacterial O2-sensors is either via altered DNA binding affinity of the sensory protein, or else due to the actions of a two-component signaling cascade. Emerging data suggests that proteins containing a hemerythrin-domain, such as FBXL5, may serve to connect iron sensing to O2-sensing in both bacteria and humans. As specific molecular machinery becomes identified, these hypoxia sensing pathways present therapeutic targets for diseases including ischemia, cancer, or bacterial infection.

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“…51 SmFixLH 128−264 and SmFixL* proteins were also characterized by MALDI-TOF mass spectrometry to confirm that the proteins were intact and pure. 44,50 Sample Preparation for PAC Measurements. Samples for PAC were prepared by diluting SmFixL* and SmFixLH 128−264 proteins into a buffer containing 20 mM Tris (pH 8.0).…”

Section: Site-directed Mutagenesis Of Smfixl* Wild-typementioning

confidence: 99%

“…42,43 Finally, Reynolds et al demonstrated the importance of the conserved, proximal Arg200 in the stabilization of the kinase inhibition related to the oxy form of SmFixL. 44 They showed a clear relation between the H-bond of Arg200 and the 6-propionate heme group that stabilized the inactive form of SmFixL upon O 2 binding to the heme.…”

Section: ■ Introductionmentioning

confidence: 99%

Photoacoustic Calorimetry Studies of O₂-Sensing FixL and (R200, I209) Variants from Sinorhizobium meliloti Reveal Conformational Changes Coupled to Ligand Photodissociation from the Heme-PAS Domain

Mokdad,

Ang,

Desciak

et al. 2023

Biochemistry

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FixL is an oxygen-sensing heme-PAS protein that regulates nitrogen fixation in the root nodules of plants. In this paper, we present the first photothermal studies of the full-length wild-type FixL protein from Sinorhizobium meliloti and the first thermodynamic profile of a full-length heme-PAS protein. Photoacoustic calorimetry studies reveal a quadriphasic relaxation for Sm FixL*WT and the five variant proteins ( Sm FixL*R200H, Sm FixL*R200Q, Sm FixL*R200E, Sm FixL*R200A, and Sm FixL*I209M) with four intermediates from <20 ns to ∼1.5 μs associated with the photodissociation of CO from the heme. The altered thermodynamic profiles of the full-length Sm FixL* variant proteins confirm that the conserved heme domain residues R200 and I209 are important for signal transduction. In contrast, the truncated heme domain, Sm FixLH 128–264 , shows only a single, fast monophasic relaxation at <50 ns associated with the fast disruption of a salt bridge and release of CO to the solvent, suggesting that the full-length protein is necessary to observe the conformational changes that propagate the signal from the heme domain to the kinase domain.

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Role of conserved Fα-helix residues in the native fold and stability of the kinase-inhibited oxy state of the oxygen-sensing FixL protein from Sinorhizobium meliloti

Cited by 6 publications

References 58 publications

Plasmodium falciparum: Nitric oxide modulates heme speciation in isolated food vacuoles

Plasmodium falciparum: Nitric oxide modulates heme speciation in isolated food vacuoles

Oxygen sensing strategies in mammals and bacteria

Photoacoustic Calorimetry Studies of O₂-Sensing FixL and (R200, I209) Variants from Sinorhizobium meliloti Reveal Conformational Changes Coupled to Ligand Photodissociation from the Heme-PAS Domain

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Role of conserved Fα-helix residues in the native fold and stability of the kinase-inhibited oxy state of the oxygen-sensing FixL protein from Sinorhizobium meliloti

Cited by 6 publications

References 58 publications

Plasmodium falciparum: Nitric oxide modulates heme speciation in isolated food vacuoles

Plasmodium falciparum: Nitric oxide modulates heme speciation in isolated food vacuoles

Oxygen sensing strategies in mammals and bacteria

Photoacoustic Calorimetry Studies of O2-Sensing FixL and (R200, I209) Variants from Sinorhizobium meliloti Reveal Conformational Changes Coupled to Ligand Photodissociation from the Heme-PAS Domain

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Photoacoustic Calorimetry Studies of O₂-Sensing FixL and (R200, I209) Variants from Sinorhizobium meliloti Reveal Conformational Changes Coupled to Ligand Photodissociation from the Heme-PAS Domain