Reactions between nitric oxide and haemoglobin under physiological conditions

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Abstract

The tenet of high-affinity nitric oxide (NO) binding to a haemoglobin (Hb) has shaped our view of haem proteins and of small diffusible signaling molecules. Specifically, NO binds rapidly to haem iron in Hb (k ≈ 107 M−1 s−1) (refs 1, 2) and once bound, the NO activity is largely irretrievable (Kd ≈ 10−5 s−1) (310); the binding is purportedly so tight as to be unaffected by O2 or CO. However, these general principles do not consider the allosteric state of Hb or the nature of the allosteric effector, and they mostly derive from the functional behaviour of fully nitrosylated Hb, whereas Hb is only partially nitrosylated in vivo11,12,13,14,15,16. Here we show that oxygen drives the conversion of nitrosylhaemoglobin in the ‘tense’ T (or partially nitrosylated, deoxy) structure to S -nitrosohaemoglobin in the ‘relaxed’ R (or ligand-bound, oxy) structure. In the absence of oxygen, nitroxyl anion (NO) is liberated in a reaction producing methaemoglobin. The yields of both S -nitrosohaemoglobin and methaemoglobin are dependent on the NO/Hb ratio. These newly discovered reactions elucidate mechanisms underlying NO function in the respiratory cycle, and provide insight into the aetiology of S -nitrosothiols, methaemoglobin and its related valency hybrids. Mechanistic re-examination of NO interactions with other haem proteins containing allosteric-site thiols may be warranted.

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Figure 1: Oxygen titration of partially nitrosylated haemoglobin.
Figure 2: Auto S -nitrosylation of haemoglobin upon oxygenation: NO group transfer from haem iron to cysteine as a function of initial NO/Hb.
Figure 3: Titration of deoxyhaemoglobin with nitric oxide: redox reactions of NO bound to the haem iron.
Figure 4: Oxygen-driven decomposition of nitrosyl haemoglobin is dependent on β-chain cysteine 93.

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Acknowledgements

We thank J. Bonaventura, D. J. Singel, H. Ishiropolous and I. Fridovich for discussion and T. McMahon for help with measurements. J.S.S. is the recipient of grants from the NHLBI; A.J.G. is supported by a National Research Award from the NHLBI.

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Correspondence to Jonathan S. Stamler.

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