Nitric Oxide Trickle Drives Heme into Hemoglobin and Muscle Myoglobin

Abstract

Ever since the days of NO being proclaimed as the “molecule of the year”, the molecular effects of this miracle gas on the globins have remained elusive. While its vasodilatory role in the cardiopulmonary system and the vasculature is well recognized, the molecular underpinnings of the NO–globin axis are incompletely understood. We show, by transwell co-culture of nitric oxide (NO) generating, HEK eNOS/nNOS cells, and K562 erythroid or C2C12 muscle myoblasts, that low doses of NO can effectively insert heme into hemoglobin (Hb) and myoglobin (Mb), making NO not only a vasodilator, but also a globin heme trigger. We found this process to be dependent on the NO flux, occurring at low NO doses and fading at higher doses. This NO-triggered heme insertion occurred into Hb in just 30 min in K562 cells and into muscle Mb in C2C12 myoblasts between 30 min and 1 h, suggesting that the classical effect of NO on upregulation of globin (Hb or Mb) is just not transcriptional, but may involve sufficient translational events where NO can cause heme-downloading into the apo-globins (Hb/Mb). This effect of NO is unexpected and highlights its significance in maintaining globins in its heme-containing holo-form, where such heme insertions might be required in the circulating blood or in the muscle cells to perform spontaneous functions.

A natural heme deficiency exists in biology that allows nitric oxide to control heme protein functions by regulating cellular heme distribution

“…Trp dioxygenase Cells and tissues [7,8,14,15] Indoleamine dioxygenase Cells [7,8,60] Hemoglobin α, β, γ Cells and tissues [23,37,71] Myoglobin Cells and tissues [23,37,71] Cytochrome P450 Cells and tissues [59] Myeloperoxidase Cells [37] NADPH oxidase 5 Cells [72] Ascorbate peroxidase a Cells [13] Horseradish peroxidase a Cells [13] a Plant enzymes were expressed in mammalian cells.…”

Hemoglobin resident in the lung epithelium is protective for smooth muscle soluble guanylate cyclase function

“…This suggests that a fine balance may exist in cell systems to maintain optimum physiological NO levels under normal healthy conditions [ 25 ]. While hsp90 and GAPDH enable heme-maturation of sGC and the globins, low NO levels can induce sGC-α1β1 heterodimerization [ 23 , 26 , 27 ] and can also trigger heme-maturation or heme-insertion directly into heme-free/apo-hemoglobin (apo-Hb) or apo-myoglobin (apo-Mb) [ 28 ] in a time frame between 30-60 min which argues against increase in globin protein synthesis. As these globin maturations are multifaceted requiring several steps and specific cell chaperons (eg.…”

Visualizing mitochondrial heme flow through GAPDH in living cells and its regulation by NO

“…However, when the NO exposure level increases the promoting effect of NO is quickly lost and instead it widely prevents cellular heme allocation to proteins [ 17 , 18 ] and can even cause some proteins to lose their heme [ 17 ]. NO displays this hormetic effect on heme allocations both when it is released from small molecule NO donors or when it is naturally generated in cells via NO synthases [ 17 , [19] , [20] , [21] ]. Overall, this has revealed a new way that NO can shape biological processes by positively or negatively regulating cell heme allocations and the consequent functions of numerous heme-dependent proteins.…”