Regulation of nitrite reductase and lipid binding properties of cytoglobin by surface and distal histidine mutations

Kaliszuk, Stefan J.; Morgan, Natasha I.; Ayers, Taylor N.; Sparacino-Watkins, Courtney; DeMartino, Anthony; Bocian, Kaitlin; Ragireddy, Venkata; Tong, Qin; Tejero, Jesús

doi:10.1016/j.niox.2022.06.001

Cited by 7 publications

(7 citation statements)

References 51 publications

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“…The Leu46Phe and Leu46Trp mutations exhibited NiR rate constants of 8.54 ± 0.25 M −1 s −1 and 0.19 ± 0.01 M −1 s −1 , respectively, showing that the phenylalanine and tryptophan mutations in the heme pocket slow NiR activity to different extents. Finally, the His81Ala mutation leads to an NiR of 1108 ± 38.0 M −1 s −1 , identical to that recently reported [ 34 ]; this is 35-fold higher in the rate of nitrite consumption compared to the WT with intramolecular disulfide, and 6500-fold higher compared to the double cysteine mutant.…”

Section: Resultssupporting

confidence: 88%

“…Replacement of the distal histidine E7 endogenous ligand by a smaller ligand (His81Ala) led to rapid biochemical activity in Cygb, supporting the gating effect of distal histidine on hexacoordinate globin, and also supporting previous studies on various globins that show that this histidine modulates the binding of the external ligand to the distal site [ 32 , 34 , 41 ]. Removal of this histidine reduces steric hindrance leading to rapid ligand binding to the distal site; the distal ligand is known to modulate ligand entry to the distal coordinate site in hemoglobins [ 35 ].…”

Section: Discussionsupporting

confidence: 74%

“…The NiR activity of Ngb was increased ~2000 times by mutation of the distal E7 histidine ligand [ 32 ]. A recent investigation into the effects of E7 mutation on Cygb NiR mutations showed varied effects ranging from 0.9 to 1100 M −1 s −1 [ 34 ]. Bimolecular ligands like NO have been shown to migrate to the distal coordination site of Hb through the rear of the heme pocket, to access the central iron.…”

Section: Introductionmentioning

confidence: 99%

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Modulating Nitric Oxide Dioxygenase and Nitrite Reductase of Cytoglobin through Point Mutations

2022

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Cytoglobin is a hexacoordinate hemoglobin with physiological roles that are not clearly understood. Previously proposed physiological functions include nitric oxide regulation, oxygen sensing, or/and protection against oxidative stress under hypoxic/ischemic conditions. Like many globins, cytoglobin rapidly consumes nitric oxide under normoxic conditions. Under hypoxia, cytoglobin generates nitric oxide, which is strongly modulated by the oxidation state of the cysteines. This gives a plausible role for this biochemistry in controlling nitric oxide homeostasis. Mutations to control specific properties of hemoglobin and myoglobin, including nitric oxide binding/scavenging and the nitrite reductase activity of various globins, have been reported. We have mapped these key mutations onto cytoglobin, which represents the E7 distal ligand, B2/E9 disulfide, and B10 heme pocket residues, and examined the nitric oxide binding, nitric oxide dioxygenase activity, and nitrite reductase activity. The Leu46Trp mutation decreases the nitric oxide dioxygenase activity > 10,000-fold over wild type, an effect 1000 times greater than similar mutations with other globins. By understanding how particular mutations can affect specific reactivities, these mutations may be used to target specific cytoglobin activities in cell or animal models to help understand the precise role(s) of cytoglobin under physiological and pathophysiological conditions.

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Section: Resultssupporting

confidence: 88%

Section: Discussionsupporting

confidence: 74%

Section: Introductionmentioning

confidence: 99%

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Modulating Nitric Oxide Dioxygenase and Nitrite Reductase of Cytoglobin through Point Mutations

2022

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“…35 Lipid Binding Experiments: The binding of sodium oleate to WT Mb and mutant Mb K46M constructs was assessed by UV-Vis spectroscopy as previously described. 36 We verified that WT Mb and Mb K46M recombinant proteins were 80-81% and 73-77% oxy-Mb, respectively, by spectra deconvolution analysis. 13 ORIGIN software (version 8) was used to fit data using Equation 1 to determine the dissociation constant (KD) as previously described.…”

Section: Generation Of Apo-mb and Mb K4m Mutants And Plasmid Expressionmentioning

confidence: 63%

Myoglobin Inhibits Breast Cancer Cell Fatty Acid Oxidation and Migration via Heme-dependent Oxidant Production and Not Fatty Acid Binding

Johnson,

Rao,

Zhang

et al. 2024

Preprint

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The monomeric heme protein myoglobin (Mb), traditionally thought to be expressed exclusively in cardiac and skeletal muscle, is now known to be expressed in approximately 40% of breast tumors. While Mb expression is associated with better patient prognosis, the molecular mechanisms by which Mb limits cancer progression are unclear. In muscle, Mb's predominant function is oxygen storage and delivery, which is dependent on the protein's heme moiety. However, prior studies demonstrate that the low levels of Mb expressed in cancer cells preclude this function. Recent studies propose a novel fatty acid binding function for Mb via a lysine residue (K46) in the heme pocket. Given that cancer cells can upregulate fatty acid oxidation (FAO) to maintain energy production for cytoskeletal remodeling during cell migration, we tested whether Mb-mediated fatty acid binding modulates FAO to decrease breast cancer cell migration. We demonstrate that the stable expression of human Mb in MDA-MB-231 breast cancer cells decreases cell migration and FAO. Site-directed mutagenesis of Mb to disrupt Mb fatty acid binding did not reverse Mb-mediated attenuation of FAO or cell migration in these cells. In contrast, cells expressing Apo-Mb, in which heme incorporation was disrupted, showed a reversal of Mb-mediated attenuation of FAO and cell migration, suggesting that Mb attenuates FAO and migration via a heme-dependent mechanism rather than through fatty acid binding. To this end, we show that Mb's heme-dependent oxidant generation propagates dysregulated gene expression of migratory genes, and this is reversed by catalase treatment. Collectively, these data demonstrate that Mb decreases breast cancer cell migration, and this effect is due to heme-mediated oxidant production rather than fatty acid binding. The implication of these results will be discussed in the context of therapeutic strategies to modulate oxidant production and Mb in tumors.

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“…27,28 Likewise the role of distal histidine has also been shown to be a key regulator in enzymatic activity of cytoglobin and doi: 10.5281/zenodo.10897345 ; Submission date: 30-03-2024 neuroglobin. [29][30][31][32] To the best of my knowledge, it must be still demonstrated whether if hemoglobin's enzymatic activity is affected by the water binding states at heme or via the distal histidine. 16 But experiments to tackle such question must take into consideration of the possibility of potential noncanonical heme binding sites as well.…”

mentioning

confidence: 99%

Heme-based water aquareceptors

Anbalagan

2024

Preprint

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How cells sense water is a fundamental question in biology and for the evolution of life. The mechanisms underlying hygrosensation in numerous organisms and animals have been experimentally demonstrated. However, hygrosensation has mainly been investigated only in specialized sensory cells (hygroreceptors). However, single-celled microorganisms must have water-sensing protein-based sensors or receptors (or aquareceptors) that would have enabled them to migrate and survive in water-scarce regions or conditions (via osmoadaptation or etc.,) and also evolve into multicellular organisms. But whether water sensing majorly occurs via osmoreceptors/osmosensors or mechanoreceptors or direct water-sensing aquareceptors is unclear. In my opinion, just as gas (solute)-sensing gasoreceptor functions in almost every cell, water-sensing aquareceptors must also function in almost every cell. Due to the potential capability of water molecules to antagonize the gas-binding sites in the heme moiety-containing sensor domains of gasoreceptors, I propose that some heme-based gas-sensing gasoreceptors may have a parallel role as water-sensing protein aquareceptors. And I wonder if hemoglobin can also be considered an aquareceptor due to its nitrite reductase activity.

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Regulation of nitrite reductase and lipid binding properties of cytoglobin by surface and distal histidine mutations

Cited by 7 publications

References 51 publications

Modulating Nitric Oxide Dioxygenase and Nitrite Reductase of Cytoglobin through Point Mutations

Modulating Nitric Oxide Dioxygenase and Nitrite Reductase of Cytoglobin through Point Mutations

Myoglobin Inhibits Breast Cancer Cell Fatty Acid Oxidation and Migration via Heme-dependent Oxidant Production and Not Fatty Acid Binding

Heme-based water aquareceptors

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