Enzymatic function of hemoglobin as a nitrite reductase that produces NO under allosteric control
Zhi Huang, Sruti Shiva, Daniel B. Kim-Shapiro, Rakesh P. Patel, Lorna A. Ringwood, Cynthia E. Irby, Kris T. Huang, Chien Ho, Neil Hogg, Alan N. Schechter, Mark T. Gladwin
Zhi Huang, Sruti Shiva, Daniel B. Kim-Shapiro, Rakesh P. Patel, Lorna A. Ringwood, Cynthia E. Irby, Kris T. Huang, Chien Ho, Neil Hogg, Alan N. Schechter, Mark T. Gladwin
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Research Article Hematology

Enzymatic function of hemoglobin as a nitrite reductase that produces NO under allosteric control

Abstract

Hypoxic vasodilation is a fundamental, highly conserved physiological response that requires oxygen and/or pH sensing coupled to vasodilation. While this process was first characterized more than 80 years ago, the precise identity and mechanism of the oxygen sensor and mediators of vasodilation remain uncertain. In support of a possible role for hemoglobin (Hb) as a sensor and effector of hypoxic vasodilation, here we show biochemical evidence that Hb exhibits enzymatic behavior as a nitrite reductase, with maximal NO generation rates occurring near the oxy-to-deoxy (R-to-T) allosteric structural transition of the protein. The observed rate of nitrite reduction by Hb deviates from second-order kinetics, and sigmoidal reaction progress is determined by a balance between 2 opposing chemistries of the heme in the R (oxygenated conformation) and T (deoxygenated conformation) allosteric quaternary structures of the Hb tetramer — the greater reductive potential of deoxyheme in the R state tetramer and the number of unligated deoxyheme sites necessary for nitrite binding, which are more plentiful in the T state tetramer. These opposing chemistries result in a maximal nitrite reduction rate when Hb is 40–60% saturated with oxygen (near the Hb P50), an apparent ideal set point for hypoxia-responsive NO generation. These data suggest that the oxygen sensor for hypoxic vasodilation is determined by Hb oxygen saturation and quaternary structure and that the nitrite reductase activity of Hb generates NO gas under allosteric and pH control.

Authors

Zhi Huang, Sruti Shiva, Daniel B. Kim-Shapiro, Rakesh P. Patel, Lorna A. Ringwood, Cynthia E. Irby, Kris T. Huang, Chien Ho, Neil Hogg, Alan N. Schechter, Mark T. Gladwin

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Sigmoidal reaction behavior of the nitrite-deoxyheme reaction occurs due...
Sigmoidal reaction behavior of the nitrite-deoxyheme reaction occurs due to T-to-R allosteric quaternary transition of Hb. (A) Model of the nitrite-deoxyHb reaction representing a balance between 2 opposing processes: (a) a decelerating reaction with T state deoxyheme due to depletion of deoxyheme available for reaction with nitrite because of conversion to metheme and iron-nitrosyl-heme (solid blue line); and (b) an accelerating reaction of nitrite with R state deoxyheme (dashed line). Solid black line with arrow represents the reaction process observed experimentally and is the balance of these 2 processes. (B) Apparent bimolecular rate constant over the time course of the anaerobic reaction of Mb (50 μM heme) and Hb (50 μM heme) with nitrite (10 mM with Hb and 2.5 mM with Mb in heme concentrations). Bimolecular rate constant (const.) was obtained by dividing the instantaneous reaction rate of deoxyheme consumption by the concentration of deoxyheme and nitrite. (C) Initial (6 minutes), intermediate (17 minutes), and final (30 minutes) EPR spectra of iron-nitrosyl-heme monitored over the course of the nitrite-deoxyHb reaction (100 μM heme, 2.5 mM nitrite) showing a transition from 5-coordinate (T state) α iron-nitrosyl-heme (with characteristic hyperfine splitting) to 6-coordinate (R state) heme geometry. The smaller EPR signals at earlier time points were normalized to that of the final time point in order to compare the spectral shape of the iron-nitrosyl-Hb signal. (D) T-to-R allosteric structural transition during the course of the nitrite-deoxyHb reaction, monitored by the percentage formation of 6-coordinate α iron-nitrosyl-heme relative to total α iron-nitrosyl-heme.