Globin X is a six-coordinate globin that reduces nitrite to nitric oxide in fish red blood cells

Abstract

The discovery of novel globins in diverse organisms has stimulated intense interest in their evolved function, beyond oxygen binding. Globin X (GbX) is a protein found in fish, amphibians, and reptiles that diverged from a common ancestor of mammalian hemoglobins and myoglobins. Like mammalian neuroglobin, GbX was first designated as a neuronal globin in fish and exhibits six-coordinate heme geometry, suggesting a role in intracellular electron transfer reactions rather than oxygen binding. Here, we report that GbX to our knowledge is the first six-coordinate globin and the first globin protein apart from hemoglobin, found in vertebrate RBCs. GbX is present in fish erythrocytes and exhibits a nitrite reduction rate up to 200-fold faster than human hemoglobin and up to 50-fold higher than neuroglobin or cytoglobin. Deoxygenated GbX reduces nitrite to form nitric oxide (NO) and potently inhibits platelet activation in vitro, to a greater extent than hemoglobin. Fish RBCs also reduce nitrite to NO and inhibit platelet activation to a greater extent than human RBCs, whereas GbX knockdown inhibits this nitrite-dependent NO signaling. The description of a novel, sixcoordinate globin in RBCs with dominant electron transfer and nitrite reduction functionality provides new insights into the evolved signaling properties of ancestral heme-globins.nitrite | nitric oxide | blood | RBC | platelet

Efficient Reduction of Vertebrate Cytoglobins by the Cytochrome b5/Cytochrome b5 Reductase/NADH System

“…Zebrafish globin X and human Ngb were expressed in the same fashion as Cygb1, as described previously 20, 26 . GbX incorporated an N-terminal His tag, and was purified by Ni-NTA agarose chromatography as described above for the Cygb proteins.…”

Evolution and expression of tissue globins in ray-finned fishes

“…More recently, multiple additional family members have been characterized that, similar to Mb, are classically considered “nonblood” or “tissue-expressed” globins [although, see Götting and Nikinmaa (2015), Corti et al. (2016a,b)], namely, neuroglobin (Ngb) (Burmester et al. 2000), cytoglobin (Cygb) (Kawada et al.…”

How Red Blood Cells Process Nitric Oxide

“…5, 6 The latter hypothesis has been supported by experimental data showing that other globins signal via nitrite reduction, including myoglobin, cytoglobin, neuroglobin and globin X. 7–9 This signaling has been shown to modulate hypoxic vasodilation, platelet activation, and mitochondrial respiration (recent platelet inhibition studies summarized 10 ). Both models propose allosteric-coupled hemoglobin deoxygenation and NO delivery and both models have central mechanisms that are testable.…”

“…5, 12 Recent studies that other globins exhibit nitrite-reductase signaling activity, confirmed with genetic knock-down approaches, provides more direct evidence in support of this hypothesis. 7–9 …”