Reply to 'Nitrite–methemoglobin inadequate for hypoxic vasodilation'

Goetz, Bradley I.; Wang, Pamela; Shields, Howard; Basu, Swati; Grubina, Rozalina; Huang, Jinming; Conradie, Jeanet; Huang, Zhi; Jeffers, Anne; Jiang, Alice; He, Xiaojun; Azarov, Ivan; Seibert, Ryan; Mehta, A. R.; Patel, Rakesh P.; King, S. Bruce; Ghosh, Abhik; Hogg, Neil; Gladwin, Mark T.; Kim‐Shapiro, Daniel B.

doi:10.1038/nchembio0609-367

Cited by 5 publications

(10 citation statements)

References 12 publications

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“…Schwab et al also showed that all of the high spin MetHb signal that disappears due to nitrite binding to MetHb can be accounted for by formation of MetHb-nitrite, affirming our assumption [29]. We therefore concluded that both methods are equivalent and that the complex behavior of nitrite binding to MetHb results in low dissociation constants (high affinity) under some conditions [30]. Here, we employ a method similar to Schwab et al to reconfirm these facets of nitrite binding to MetHb.…”

Section: Resultssupporting

confidence: 84%

“…Schwab et al obtained a similar dissociation constant (1.8 mM) testing a range of nitrite:MetHb ratios in Hepes buffer at pH 7.4 [29]. These values are much higher than those we have reported under some conditions [24; 30] and those obtained here. We hold that rather than involving simple reaction, with a single association and dissociation rate, nitrite binding to MetHb is complex and may play important roles in nitrogen oxide biology.…”

Section: Discussionsupporting

confidence: 75%

“…Based on analysis of EPR data, we reported that the affinity of nitrite for MetHb is much higher under some conditions than has been reported previously [24]. The methods used in the EPR experiments and conclusions drawn have been challenged [29] and we have presented a short, formal reply arguing that our original methods and conclusions are valid [30]. …”

Section: Introductionmentioning

confidence: 93%

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An electron paramagnetic resonance study of the affinity of nitrite for methemoglobin

et al. 2010

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Recent data suggests that reactions of nitrite with ferric hemoglobin are potentially important in heme-protein dependent NO signaling. Our group and others are evaluating the role of reductive nitrosylation reactions in the generation of N2O3 as a signaling molecule. The latter reaction is hypothesized to involve reactions on NO, nitrite and methemoglobin to form N2O3 in a anydrase reaction. Of potential importance to these reactions is the affinity of methemoglobin for nitrite and the reactivity of nitrite bound methemoglobin with nitric oxide. In this paper we review work related to the electronic structure of nitrite bound methemoglobin and its dissociation constant. We present new data using electron paramagnetic resonance spectroscopy which confirm that methemoglobin has a much higher affinity for nitrite, under certain conditions, than reported in classical observations. Interestingly the affinity is greatest at lower pH and low nitrite:methemoglobin ratios. These data suggest additional interesting chemistry in the reaction of nitrite with ferric and ferrous heme species. Moreover, this reaction could serve as a paradigm for ferric heme reactions with nitrite.

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Section: Resultssupporting

confidence: 84%

Section: Discussionsupporting

confidence: 75%

Section: Introductionmentioning

confidence: 93%

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An electron paramagnetic resonance study of the affinity of nitrite for methemoglobin

et al. 2010

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“…1, bottom). Nevertheless the fundamental features are the same; intrinsic buffer effects represent a modest perturbation, in contrast to suggestions made by others 18 .…”

Section: Resultsmentioning

confidence: 56%

“…The assayed concentrations confirmed mass balance of protein before and after the addition of nitrite; no evidence for formation of EPR silent products was observed. The concentration of nitrite in solution was calculated as original nitrite added less metHb:NO 2 − formed, as done by others 6, 18, 24 . The absence of change in the metHb:NO 2 − EPR spectrum over several hours suggests that there is no or limited consumption of nitrite under our reaction conditions, supporting the method employed to estimate nitrite concentration.…”

Section: Resultsmentioning

confidence: 99%

EPR Spectroscopy of Nitrite Complexes of Methemoglobin

2010

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The chemical interplay of nitrogen oxides (NO's) with hemoglobin (Hb) has attracted considerable recent attention because of its potential significance in the mechanism of NO-related vasoactivity regulated by Hb. An important theme of this interplayredox coupling in adducts of heme iron and NO'shas sparked renewed interest in fundamental studies of FeNO x coordination complexes. In this Article, we report combined UV−vis and comprehensive electron paramagnetic resonance (EPR) spectroscopic studies that address intriguing questions raised in recent studies of the structure and affinity of the nitrite ligand in complexes with FeIII in methemoglobin (metHb). EPR spectra of metHb/NO2 − are found to exhibit a characteristic doubling in their sharper spectral features. Comparative EPR measurements at X- and S-band frequencies, and in D2O versus H2O, argue against the assignment of this splitting as hyperfine structure. Correlated changes in the EPR spectra with pH enable complete assignment of the spectrum as deriving from the overlap of two low-spin species with g values of 3.018, 2.122, 1.45 and 2.870, 2.304, 1.45 (values for samples at 20 K and pH 7.4 in phosphate-buffered saline). These g values are typical of g values found for other heme proteins with N-coordinated ligands in the binding pocket and are thus suggestive of N-nitro versus O-nitrito coordination. The positions and shapes of the spectral lines vary only slightly with temperature until motional averaging ensues at ∼150 K. The pattern of motional averaging in the variable-temperature EPR spectra and EPR studies of FeIIINO2 −/FeIINO hybrids suggest that one of two species is present in both of the α and β subunits, while the other is exclusive to the β subunit. Our results also reconfirm that the affinity of nitrite for metHb is of millimolar magnitude, thereby making a direct role for nitrite in physiological hypoxic vasodilation difficult to justify.

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