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Nitrite binding to globins: linkage isomerism, EPR silence and reductive chemistry.

Silaghi-Dumitrescu R, Svistunenko DA, Cioloboc D, Bischin C, Scurtu F, Cooper CE - Nitric Oxide (2014)

Bottom Line: We have used EPR (electron paramagnetic resonance) and DFT (density functional theory) to explore these binding modes to myoglobin and hemoglobin.The EPR and DFT data show that both nitrite linkage isomers can be present at the same time and that the two isomers are readily interconvertible in solution.The millisecond-scale process of nitrite reduction by Hb is investigated in search of the elusive Fe(II)-nitrite adduct.

View Article: PubMed Central - PubMed

Affiliation: "Babeş-Bolyai" University, 1 Mihail Kogalniceanu str., RO-400084 Cluj-Napoca, Romania; Department of Biological Sciences, University of Essex, Wivenhoe Park, Colchester, Essex CO4 3SQ, UK. Electronic address: rsilaghi@chem.ubbcluj.ro.

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Upper panel: left UV-vis spectra of deoxyhemoglobin treated with nitrite, recorded for up to 2 seconds after mixing. Conditions: deoxyhemoglobin was obtained by purging with argon, and then adding a glucose oxidaze/glucose/catalase mixture as indicated in Experimental, buffer PBS, pH = 7.4, [Hb] = 18 µM, [nitrite] = 925 mM. Right: involved species as resulted from the fitted spectra, employing the A → B, B → C model. Lower panel: left: time evolution of the three fitted species, A, B and C; right: overlay of kinetic data and of fitted trace at some representative wavelength.
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f0040: Upper panel: left UV-vis spectra of deoxyhemoglobin treated with nitrite, recorded for up to 2 seconds after mixing. Conditions: deoxyhemoglobin was obtained by purging with argon, and then adding a glucose oxidaze/glucose/catalase mixture as indicated in Experimental, buffer PBS, pH = 7.4, [Hb] = 18 µM, [nitrite] = 925 mM. Right: involved species as resulted from the fitted spectra, employing the A → B, B → C model. Lower panel: left: time evolution of the three fitted species, A, B and C; right: overlay of kinetic data and of fitted trace at some representative wavelength.

Mentions: Fig. 6 shows the UV-vis spectra collected during the reaction of deoxyhemoglobin with excess nitrite. The first spectrum, characterized by a band with a maximum at 552 nm, is attributable to the starting deoxyhemoglobin, while the last one, characterized by three absorption maxima at 502, 531 and 590 nm, is a mixture of the met-nitrite adduct with the Fe(II)-NO (Supporting material Fig. S2), findings in concordance with other studies [12]. The EPR spectrum of species C confirms the presence of the Fe(II)-NO adduct, even if the superhiperfine coupling is not completed resolved (Supporting material Fig. S3). The intermediate, species B in Fig. 6, bears excellent resemblance to the ferric-NO adduct. The implication is that, en route from deoxy to met-NO, the ferrous-nitrosyl is formed at a rate significantly slower than its decay, and that it, therefore, does not accumulate to a level detectable by UV-vis spectroscopy even under large excess of nitrite.


Nitrite binding to globins: linkage isomerism, EPR silence and reductive chemistry.

Silaghi-Dumitrescu R, Svistunenko DA, Cioloboc D, Bischin C, Scurtu F, Cooper CE - Nitric Oxide (2014)

Upper panel: left UV-vis spectra of deoxyhemoglobin treated with nitrite, recorded for up to 2 seconds after mixing. Conditions: deoxyhemoglobin was obtained by purging with argon, and then adding a glucose oxidaze/glucose/catalase mixture as indicated in Experimental, buffer PBS, pH = 7.4, [Hb] = 18 µM, [nitrite] = 925 mM. Right: involved species as resulted from the fitted spectra, employing the A → B, B → C model. Lower panel: left: time evolution of the three fitted species, A, B and C; right: overlay of kinetic data and of fitted trace at some representative wavelength.
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Related In: Results  -  Collection

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getmorefigures.php?uid=PMC4256065&req=5

f0040: Upper panel: left UV-vis spectra of deoxyhemoglobin treated with nitrite, recorded for up to 2 seconds after mixing. Conditions: deoxyhemoglobin was obtained by purging with argon, and then adding a glucose oxidaze/glucose/catalase mixture as indicated in Experimental, buffer PBS, pH = 7.4, [Hb] = 18 µM, [nitrite] = 925 mM. Right: involved species as resulted from the fitted spectra, employing the A → B, B → C model. Lower panel: left: time evolution of the three fitted species, A, B and C; right: overlay of kinetic data and of fitted trace at some representative wavelength.
Mentions: Fig. 6 shows the UV-vis spectra collected during the reaction of deoxyhemoglobin with excess nitrite. The first spectrum, characterized by a band with a maximum at 552 nm, is attributable to the starting deoxyhemoglobin, while the last one, characterized by three absorption maxima at 502, 531 and 590 nm, is a mixture of the met-nitrite adduct with the Fe(II)-NO (Supporting material Fig. S2), findings in concordance with other studies [12]. The EPR spectrum of species C confirms the presence of the Fe(II)-NO adduct, even if the superhiperfine coupling is not completed resolved (Supporting material Fig. S3). The intermediate, species B in Fig. 6, bears excellent resemblance to the ferric-NO adduct. The implication is that, en route from deoxy to met-NO, the ferrous-nitrosyl is formed at a rate significantly slower than its decay, and that it, therefore, does not accumulate to a level detectable by UV-vis spectroscopy even under large excess of nitrite.

Bottom Line: We have used EPR (electron paramagnetic resonance) and DFT (density functional theory) to explore these binding modes to myoglobin and hemoglobin.The EPR and DFT data show that both nitrite linkage isomers can be present at the same time and that the two isomers are readily interconvertible in solution.The millisecond-scale process of nitrite reduction by Hb is investigated in search of the elusive Fe(II)-nitrite adduct.

View Article: PubMed Central - PubMed

Affiliation: "Babeş-Bolyai" University, 1 Mihail Kogalniceanu str., RO-400084 Cluj-Napoca, Romania; Department of Biological Sciences, University of Essex, Wivenhoe Park, Colchester, Essex CO4 3SQ, UK. Electronic address: rsilaghi@chem.ubbcluj.ro.

Show MeSH
Related in: MedlinePlus