Nitrite binding to globins: linkage isomerism, EPR silence and reductive chemistry.
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.
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: firstname.lastname@example.org.Show MeSH
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Mentions: UV-vis and EPR spectra of the nitrite adduct of ferric human hemoglobin are shown in Fig. 3. The optical spectra suggest that Hb features a ~5-fold increase in affinity toward nitrite compared with Mb (3 mM vs. 14 mM); other hemoglobins (cow, horse, dog, rat, sheep – data not shown) likewise show similar affinities, and clear evidence of cooperativity was not found in any of them. The apparent EPR-silence observed in the previous reports  for the Hb-nitrate adduct was later interpreted as a saturation of the low spin species at a lower microwave power . Our data are in agreement with others citing where the EPR signal due to the Hb-nitrate signal is partially visible with some dependence on the buffering agent. Although the signal is smaller than expected they are visible at g~6 and g~3 (similar to the Mb-nitrite adduct), suggesting that linkage isomerism is also at work in Hb. Furthermore, two sets of low-spin signals are seen for the nitrite adduct (3.03/2.33 and 2.90/2.17), which suggests that at least two possible distinct conformations are available for the Fe-NO2− moiety; these two conformations are likely possible on the same subunit as opposed to being restricted on the α and β subunits, respectively, since the Fig. 2 Mb-nitrite EPR also shows some asymmetry in the g~3 signal and since there seems to be a concentration and pH dependence on their relative distributions (cf. Supporting material). Also, the experiments performed at 100°K, in concordance with the results obtained by Goetz et al.  (Supplementary material Fig. S1), show a direct correlation between the square root of the power applied and the intensity of the g = 6 for MetHb, as well as for MetHb-nitrate.Figs. 2 and 3 also reveal a small difference between the electronic absorption UV-vis spectra of the Hb and Mb nitrite adducts, with the 530–580 nm features, specific to low-spin heme, being more pronounced in hemoglobin than in myoglobin. Interestingly, this difference is mirrored by the EPR spectra, where addition of nitrite causes the g~6 signal to decrease distinctly more in Hb than in Mb.
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: email@example.com.