Effects of T- and R-state stabilization on deoxyhemoglobin-nitrite reactions and stimulation of nitric oxide signaling

Abstract

Recent data suggest that transitions between the relaxed (R) and tense (T) state of hemoglobin control the reduction of nitrite to nitric oxide (NO) by deoxyhemoglobin. This reaction may play a role in physiologic NO homeostasis and be a novel consideration for the development of the next generation of hemoglobin-based blood oxygen carriers (HBOCs, i.e. artificial blood substitutes). Herein we tested the effects of chemical stabilization of bovine hemoglobin in either the T-(THb) or R-state (RHb) on nitrite reduction kinetics, NO-gas formation and ability to stimulate NOdependent signaling. These studies were performed over a range of fractional saturations that is expected to mimic biological conditions. The initial rate for nitrite-reduction decreased in the following order RHb > bHb > THb, consistent with the hypothesis that the rate constant for nitrite reduction is faster with R-state Hb and slower with T-state Hb. Moreover, RHb produced more NO-gas and inhibited mitochondrial respiration more potently than both bHb and THb. Interestingly, at low oxygen fractional saturations, THb produced more NO and stimulated nitritedependent vasodilation more potently than bHb despite both derivatives having similar initial rates for nitrite reduction and a more negative reduction potential in THb versus bHb. These data suggest that cross-linking of bovine hemoglobin in the T-state conformation leads to a more effective coupling of nitrite reduction to NO-formation. Our results support the model of allosteric regulation of nitrite reduction by deoxyhemoglobin and show that cross-linking hemoglobins in Publisher's Disclaimer: This is a PDF file of an unedited manuscript that has been accepted for publication. As a service to our customers we are providing this early version of the manuscript. The manuscript will undergo copyediting, typesetting, and review of the resulting proof before it is published in its final citable form. Please note that during the production process errors may be discovered which could affect the content, and all legal disclaimers that apply to the journal pertain. NIH Public Access

HbE/β-Thalassemia and Oxidative Stress: The Key to Pathophysiological Mechanisms and Novel Therapeutics

“…NO production via NR is most effective when there is a substantial population of deoxyheme. Optimum production appears to occur for partially oxygenated Hb that is stabilized in the R state quaternary conformation (29,83,94,199). Under these conditions, the resulting NO is likely to be captured by vacant deoxyheme sites to produce nitrosylated hemes (e.g., HbNO), which are very stable.…”

Inhalation of NO during myocardial ischemia reduces infarct size and improves cardiac function

“…In murine and ovine models of ischemic stroke, we recently realized this effect through inhalation of NO [5]. Inhaled NO (NOi) results in the formation of stable NO carriers in blood, namely nitrite [6,7], which may conserve and circulate NO bioactivity and release NO when hemoglobin desaturates [8][9][10], thus redistributing blood flow preferentially to hypoxic tissues. Hence, we hypothesized that NOi may provide a novel and attractive therapeutic option for the treatment of AMI, and tested this concept in a rat model of AMI.…”

Generating S-Nitrosothiols from Hemoglobin

“…Furthermore, the rate of the reaction is allosterically controlled. Studies on partially oxygenated Hb (32,49), chemically modified Hbs (31,53,54), sol-gel-encapsulated Hbs (55), and Hb dimers bound to haptoglobin (50) all indicate the R state deoxy-hemes manifest a faster initial rate for this reaction by a factor of approximately 10 over the corresponding T state deoxy-hemes. R and T states refer here, respectively, to the high and low oxygen affinity states associated with the quaternary structures of the fully oxygenated and fully deoxygenated derivatives of Hb.…”

“…This allosterically responsive behavior links this reaction with hypoxic vasodilation. As part of the mechanism, a half-oxygenated R state Hb would have the maximum NO-generating efficacy in the NR reaction (32,49,53) because it represents a compromise between accessible reactive deoxy-heme sites and the higher reactivity associated with the R state (i.e. too much deoxygenation results in T state formation and too much oxygenation results in too few sites available for the reaction with nitrite).…”