The reactions between nitrite and hemoglobin have been studied for over a century [13
]. The recent interest in this reaction stems from its potential to be a source of NO in vivo in vascular beds where hemoglobin deoxygenation occurs. This paradigm may be important in physiologic mechanisms for regulation of hypoxic blood flow [3
], in the therapeutic mechanisms for nitrite mediated protection against ischemia induced injury [53
] and more recently, dysfunction in this reaction has been implicated in the pathogenesis of human sepsis [54
]. Data presented herein further support the concept that deoxyhemoglobin-nitrite reactions are a source of NO that activates NO-dependent signaling pathways.
Nitrite reduction by deoxyhemoglobin is now appreciated to be regulated by protein conformation with the reaction of R-state being faster than T-state [11
]. Additionally, vacant ferrous hemes are required for nitrite reduction by hemoglobin. These properties result in a bell-shaped dependence for the initial rate (the product of both nitrite and deoxyheme concentration and the rate constant) of nitrite reduction as a function of fractional saturation with maximal rates observed around the P50
]. Data presented herein using native bovine Hb that can undergo T to R transitions, and cross-linked polymeric bovine hemoglobins that have been stabilized in either T- or R-state conformation provide further evidence for this model. With native hemoglobin, the initial rate for nitrite-mediated deoxyhemoglobin consumption exhibited a bell-shaped dependence on fractional saturation, albeit not as pronounced as previously reported with human hemoglobin [4
] which possibly reflects species-species variation and requires further investigation. Both RHb and THb showed linear dependent increases in initial rate for nitrite reduction as a function of decreasing fractional saturation consistent with these molecules being conformationally locked and unable to undergo allosteric transition. Also consistent with the proposed model, nitrite reduction was fastest with RHb and slowest with THb. In this model however, it would be expected then that the initial rate for nitrite reduction by bHb at higher fractional saturations (predominantly R-state) would be similar to RHb. However three-fold lower concentrations of nitrite were required in order to follow deoxyhemoglobin consumption kinetics for the latter derivative, indicating a faster rate of reaction. Both a more negative redox potential and increased heme accessibility have been proposed to mediate increased nitrite reduction rates by R-deoxy vs. T-deoxy hemoglobin [55
]. The lack of data regarding the redox potential of RHb due to technical limitations in our experimental setup precludes us from formulating an hypothesis in this regard but we note that reports exist in the literature suggesting that hemoglobin cross-linking leads to increased heme accessibility [16
] thereby potentially resulting in increased reactivity towards nitrite. In addition, we cannot exclude the possibility that even at high fractional saturations, the presence of low levels of T-state hemes in bHb versus exclusively R-state hemes in RHb, result in a lower rate of nitrite reduction.
Unlike RHb, THb showed slower initial rates compared to bHb across most fractional saturations. Moreover at low fractional saturations where native hemoglobin will be in the T-state, initial rates for native Hb and THb are similar. Interestingly, the reduction potential of THb was lower than that of bHb suggesting that increased rates of nitrite reduction should have been observed (see ) if electron exchange with the heme was the rate-limiting step. The fact that no differences in nitrite reduction kinetics were observed under lower fractional saturations suggests that polymerization of T-state hemoglobin may decrease heme accessibility and that this plays an important role in regulating nitrite-reduction. As indicated above, at intermediate fractional saturations bHb will be comprised of both R- and T-state hemes compared to only R- or T-heme in RHb and THb respectively which in turn may affect nitrite reduction kinetics. At a fractional saturation of zero however, where all hemes in bHb and THb are in T-state, differences in nitrite reduction kinetics were not observed despite differences in redox potential. Under these conditions, heme accessibility differences are the likeliest explanation. We also note that this behavior underscores the importance of assessing nitrite reduction kinetics across fractional saturations as determination of this parameter under anoxic conditions (fractional saturation of 0) alone would have suggested that THb and bHb were similar.
Recent studies have questioned whether free NO is formed at all during hemoglobin-nitrite reactions [56
]. Formation of NO-gas and the ability of nitrite-derived NO to inhibit mitochondrial respiration and stimulate vasodilation were determined therefore to assess first if different hemoglobins could reduce nitrite to NO and then, whether this NO was competent to elicit two independent signaling responses. This also allowed evaluation of how kinetics of deoxyhemoglobin consumption translated into NO-formation and bioactivity. Anoxic NO-gas formation was highest with RHb, being ~5 fold greater than native Hb and consistent with the differences observed in deoxyhemoglobin consumption kinetics. Unexpectedly however, THb produced more NO compared to native Hb despite similar deoxyhemoglobin consumption kinetics at zero fractional saturation (See ). Data from mitochondrial inhibition experiments showed that the degree of inhibition by hemoglobins and nitrite mirrored NO-gas formation profiles suggesting that bioactive NO is formed from all the hemoglobins; and furthermore, that NO-gas formation under anoxia was a better predictor for activation of NO-signaling than deoxyhemoglobin consumption rates. Vessel relaxation studies also suggested more nitrite-derived NO from THb relative to bHb. The discordance between THb consumption kinetics and NO-formation / signaling suggests that THb more effectively couples nitrite reduction to NO-generation. Recent insights into NO-formation from nitrite-hemoglobin reactions suggest roles for oxidative denitrosylation of nitrosylhemoglobin [30
] and formation of a dinitrogen trioxide (N2
) intermediate which can homolyze to NO and nitrogen dioxide (NO2
). In this model, nitrite binding to ferric hemoglobin first forms a ferrous-NO2
radical intermediate that in turn reacts with NO to form N2
]. These possibilities coupled with other observations showing that nitrite binds to heme via an O-binding mode [58
] illustrates the complexity, and potential for multiple reactions that occur to produce NO from nitrite-hemoglobin interactions. It is not clear how T-state polymerization affects these reactions but it is possible that differential effects on any one could result in more efficient NO-production and such information could be useful in the development of HBOCs. Clearly, these data further underscore the importance of assessing functional signaling end points in comparing different hemoglobins and their reactions with nitrite to generate NO.
HBOC mediated scavenging of NO has received considerable interest recently, with clinical development of these products being halted due to potential detrimental effects of NO-scavenging [27
]. Nitrite represents a potential adjuvant therapy for HBOC with the concept being that as HBOC deoxygenate in respiring tissues, they would reduce nitrite to form NO and both increase blood flow and replete NO-signaling in general. These concepts are illustrated by data from vessel relaxation experiments which show that hemoglobin, irrespective of it being native or T-state stabilized, inhibits NO-dependent vasodilation to similar extents and consistent with similar rate constants for NO dioxygenation by bHb and THb (). Notably, we observed that both THb and bHb were 2-fold more efficient at inhibiting MNO-mediated vasorelaxation at low versus high fractional saturations (). This result was unexpected since NO-scavenging rates for oxy- and deoxy hemes, are similar being in fact slightly higher for oxyhemoglobin. We observed MNO itself was ~2.3 times more potent at inducing vasorelaxation at high versus low oxygen tensions (data not shown) suggesting that the efficacy of NO-soluble guanylate cyclase (sGC) activation signaling pathway is decreased at low oxygen tension. If we consider that the inhibition of NO-mediated vasorelaxation by hemoglobin responds to a competition between hemoglobin and sGC, then as the oxygen tension is lowered the decrease in reactivity between sGC and NO will result in a increased hemoglobin mediated inhibition at lower fractional saturations. In the case of nitrite, both THb and bHb inhibit vasodilatation at high fractional saturations. Previous data have shown that in the absence of added hemoglobin, nitrite promotes vasodilation via NO-formation at high and low oxygen tensions [21
]. At high oxygen tensions therefore, native- and THb-dependent inhibition of nitrite-mediated vasorelaxation is likely due to the NO-dioxygenation reaction. However, as the fractional saturation of the hemoglobins is lowered, the inhibitory effect on nitrite dependent vasodilation is lost and eventually transitions into a potentiation of the response. Importantly, the overall effect on vascular tone should be considered as a balance between both hemoglobin-mediated vasoconstriction (NO-scavenging) and vasodilation (nitrite-reduction to NO). Since NO-scavenging occurs across the entire fractional saturation range, the data with nitrite support the model where as hemoglobin deoxygenates, more NO is made from nitrite which counters NO-scavenging reactions eventually shifting to net NO-formation. Interestingly, this counter balancing effect of nitrite reduction was more potent with THb vs native Hb as indicated by an ~2-fold steeper gradient for percent inhibition versus fractional saturation relationship () which also mirrors increased NO-formation from THb.
In summary, data presented support the model that allosteric regulation of deoxyhemoglobin-mediated nitrite reduction can yield bioactive NO. Moreover, data show that hemoglobin cross-linking and conformational locking can dramatically impact on nitrite-reduction kinetics, NO-formation and bioactivity thereby resulting in hemoglobins that can more effectively couple nitrite-reduction to stimulation of NO-bioactivity. Finally, these data also show that nitrite reduction kinetics measured by deoxyhemoglobin consumption do not necessarily reflect the yield of NO. In this regard, the determination of NO gas production and assessment of NO-dependent signaling provides a more accurate indicator of the ability of different hemoglobins to produce bioactive NO from nitrite reduction. This observation might be of particular relevance when assessing the ability of different HBOCs to function as nitrite reductases under physiological conditions.