EPR Spectroscopy of Nitrite Complexes of Methemoglobin

Abstract

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.

A biosensor platform for soil management: the case of nitrites

“…At pH values above 6.5 sensitivity towards nitrites diminished; the signal corresponded to 62.5 mg/L nitrite ion (in bulk electrolyte) reduced to 48% at pH 7, 18.8% at pH 7.5, and to noise levels at pH 8.0. The results are consistent with previous reports on the methemoglobinenitrite complex (Goetz et al, 2010;Schwab et al, 2010).…”

“…The ammonium-N ðN À NH þ 4 Þ content was measured by the silicate-hypochlorite method (Bower and Holm-Hansen, 1980); the Strickland and Heme proteins have a known affinity for nitrite (Rodkey, 1976;Unnikrishnan and Rao, 1992;Iwoha and Okonjo, 1993;Laterreur and English, 2007), although the formation of the complex has not been fully elucidated yet. The electron paramagnetic resonance analysis of Schwab et al (2010) indicated a low affinity for nitrites, whereas Goetz et al (2010) reported a higher affinity under certain conditions: low pH values and nitrite:methemoglobin ratios. The hemoglobinenitrite interaction exhibits complex kinetics, having a lag phase, an autocatalytic step and concentration-dependant inhibition (Rodkey, 1976), resulting in long response times (6.7 min, prolonged to 45 min by hyperoxide scavengers) and low selectivity (Unnikrishnan and Rao, 1992).…”

“…Many studies suggest that at pH values > 7.7, nitrite pushes methemoglobin towards the relaxed (R)-state (Rodkey, 1976;Iwoha and Okonjo, 1993;Schwab et al, 2010) resulting in high dissociation constants (Goetz et al, 2010); at pH 6.5, methemoglobin is at the tense (T)-state exhibiting a high affinity for nitrite and a very low dissociation constant that becomes significant only in high millimolar nitrite levels (Goetz et al, 2010).…”

“…The charge on methemoglobin becomes progressively more negative on membranes as nitrite concentration increases (Goetz et al, 2010;Schwab et al, 2010), leading to an increase of the concentration of cations in the vicinity of the bilayer. The result could be a rapid increase of ion flux through the bilayer, observed as higher steady-state current values (Siontorou and Nikolelis, 1997).…”

Preclinical Evaluation of the Metabolism and Disposition of RRx-001, a Novel Investigative Anticancer Agent

“…A direct effect from the conversion of RRx-001 to NO X is possible given the demonstrated loss of the nitro groups both on reaction with GSH and after alkylation to Hb. There are many enzymatic systems involved in the reduction of nitrite to nitric oxide (van Faassen et al, 2009;Tiso et al, 2011), and the pharmacokinetics of NO X is complex (Hon et al, 2010;Stamler and Hess, 2010;Seth and Stamler, 2011;Schwab et al, 2010).…”