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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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Top panel: UV-vis spectra of ferric myoglobin (100 µM) in the presence of varying amounts of nitrite, in 50 mM phosphate pH 7.4, room temperature; shown as inset is a binding curve whose theoretical fit indicates a binding constant of 14 mM. Bottom panel: EPR spectra of Mb in the presence and absence of nitrite, at 10 K. The g-values of the signals are indicated. Instrument conditions: microwave frequency 9.47 GHz, microwave power 3.18 mW, modulation frequency 100 kHz, modulation amplitude 5 G, sweep rated 22.6 G/s; time constant 81.92 ms, single sweep for each spectrum.
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f0020: Top panel: UV-vis spectra of ferric myoglobin (100 µM) in the presence of varying amounts of nitrite, in 50 mM phosphate pH 7.4, room temperature; shown as inset is a binding curve whose theoretical fit indicates a binding constant of 14 mM. Bottom panel: EPR spectra of Mb in the presence and absence of nitrite, at 10 K. The g-values of the signals are indicated. Instrument conditions: microwave frequency 9.47 GHz, microwave power 3.18 mW, modulation frequency 100 kHz, modulation amplitude 5 G, sweep rated 22.6 G/s; time constant 81.92 ms, single sweep for each spectrum.

Mentions: Fig. 2 shows UV-vis and EPR spectra of ferric myoglobin in the presence of nitrite, at acidic and basic pH. The optical spectra indicate a binding constant of 14 mM for nitrite on Mb. The EPR spectra are thus recorded at full saturation (200 mM) and should only feature signals due to the nitrite-Mb adduct; it is thus seen that nitrite induces a low-spin signal which, due to its g~3 feature, is most likely attributable to a nitrogenous ligand [39]. This suggests that, contrary to what is seen in the crystal structure, in solution nitrite can in fact bind to the iron in myoglobin via its nitrogen atom. However, the area under this signal accounts for only 44% of the heme present in the sample [20]. A high-spin g~6 signal is also observed in the nitrite-treated sample, suggestive of a high-spin ferric state, accounting to 14% of the heme. In view of the optical titration data the aqua-like g~6 signal seen in the presence of 200 mM nitrite must be attributed to a nitrite adduct; due to the similarity in shape with the aqua signal, this nitrite g~6 form is most easily attributed as due to nitrite binding to iron via its oxygen atom. To our knowledge the g~6 signal in globins is largely unaffected by the identity of the axial ligand, so that it would not be unexpected for a nitrite adduct to feature a signal similar to that of the aqua.


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)

Top panel: UV-vis spectra of ferric myoglobin (100 µM) in the presence of varying amounts of nitrite, in 50 mM phosphate pH 7.4, room temperature; shown as inset is a binding curve whose theoretical fit indicates a binding constant of 14 mM. Bottom panel: EPR spectra of Mb in the presence and absence of nitrite, at 10 K. The g-values of the signals are indicated. Instrument conditions: microwave frequency 9.47 GHz, microwave power 3.18 mW, modulation frequency 100 kHz, modulation amplitude 5 G, sweep rated 22.6 G/s; time constant 81.92 ms, single sweep for each spectrum.
© Copyright Policy
Related In: Results  -  Collection

Show All Figures
getmorefigures.php?uid=PMC4256065&req=5

f0020: Top panel: UV-vis spectra of ferric myoglobin (100 µM) in the presence of varying amounts of nitrite, in 50 mM phosphate pH 7.4, room temperature; shown as inset is a binding curve whose theoretical fit indicates a binding constant of 14 mM. Bottom panel: EPR spectra of Mb in the presence and absence of nitrite, at 10 K. The g-values of the signals are indicated. Instrument conditions: microwave frequency 9.47 GHz, microwave power 3.18 mW, modulation frequency 100 kHz, modulation amplitude 5 G, sweep rated 22.6 G/s; time constant 81.92 ms, single sweep for each spectrum.
Mentions: Fig. 2 shows UV-vis and EPR spectra of ferric myoglobin in the presence of nitrite, at acidic and basic pH. The optical spectra indicate a binding constant of 14 mM for nitrite on Mb. The EPR spectra are thus recorded at full saturation (200 mM) and should only feature signals due to the nitrite-Mb adduct; it is thus seen that nitrite induces a low-spin signal which, due to its g~3 feature, is most likely attributable to a nitrogenous ligand [39]. This suggests that, contrary to what is seen in the crystal structure, in solution nitrite can in fact bind to the iron in myoglobin via its nitrogen atom. However, the area under this signal accounts for only 44% of the heme present in the sample [20]. A high-spin g~6 signal is also observed in the nitrite-treated sample, suggestive of a high-spin ferric state, accounting to 14% of the heme. In view of the optical titration data the aqua-like g~6 signal seen in the presence of 200 mM nitrite must be attributed to a nitrite adduct; due to the similarity in shape with the aqua signal, this nitrite g~6 form is most easily attributed as due to nitrite binding to iron via its oxygen atom. To our knowledge the g~6 signal in globins is largely unaffected by the identity of the axial ligand, so that it would not be unexpected for a nitrite adduct to feature a signal similar to that of the aqua.

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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Related in: MedlinePlus