Slow Ligand Binding Kinetics Dominate Ferrous Hexacoordinate Hemoglobin Reactivities and Reveal Differences between Plants and Other Species

Abstract

Hexacoordinate hemoglobins are found in many living organisms ranging from prokaryotes to plants and animals. They are named “hexacoordinate” because of reversible coordination of the heme iron by a histidine side chain located in the heme pocket. This endogenous coordination competes with exogenous ligand binding and causes multiphasic relaxation time courses following rapid mixing or flash photolysis experiments. Previous rapid mixing studies have assumed a steady-state relationship between hexacoordination and exogenous ligand binding that does not correlate with observed time courses for binding. Here, we demonstrate that this assumption is not valid for some hexacoordinate hemoglobins, and that multiphasic time courses are due to an appreciable fraction of pentacoordinate heme resulting from relatively small equilibrium constants for hexacoordination (K H). CO binding reactions initiated by rapid mixing are measured for four plant hexacoordinate hemoglobins, human neuroglobin and cytoglobin, and Synechocystis hemoglobin. The plant proteins, while showing a surprising degree of variability, differ from the others in having much lower values of K H. Neuroglobin and cytoglobin display dramatic biphasic time courses for CO binding that have not been observed using other techniques. Finally, an independent spectroscopic quantification of K H is presented that complements rapid mixing for the investigation of hexacoordination. These results demonstrate that hexacoordination could play a much larger role in regulating affinity constants for ligand binding in human neuroglobin and cytoglobin than in the plant hexacoordinate hemoglobins.

Characterization of a Globin-coupled Oxygen Sensor with a Gene-regulating Function

“…The CO binding behavior of AvGReg mirrors the close similarity between the absorption spectrum in the Soret region of the deoxygenated derivative of the AvGReg hemoprotein (Fig. 2B) and that of hexa-coordinate bis-histidyl adducts (27,87,88). A simple mechanism involving only the hexa-to penta-coordination of the heme-Fe atom step preceding the AvGReg hemoprotein carbonylation process does not fit the experimental data (data not shown), because it cannot account for the very slow process, characterized by k 1 and k Ϫ1 (Fig.…”

Iron Bioavailability of Maize Hemoglobin in a Caco-2 Cell Culture Model

“…Maize globin was expressed in E. coli, purified, and characterized as described by Smagghe et al 37 The protein was purified using metal affinity chromatography, resulting in a single band on an SDS-PAGE gel, and had a Soret/280 ratio that was characteristic of pure hemoglobin. 37 For Figure 1. Gene construct used to create maize overexpressing maize globin.…”

Structure and Reactivity of Hexacoordinate Hemoglobins

“…The two other vertebrate hxHbs, "neuroglobins" (Ngb) and "globin X" (GlbX), are more closely related to extracellular oxygen transport Hbs present in animals with closed circulatory systems [19,43,44]. In general, the Ngbs are very strongly coordinated by the distal histidine in both the ferrous and ferric oxidation states, Cgbs are intermediate in this regard, and hxHbs from branch 1 of the tree are most weakly hexacoordinate, on par with the nsHbs from plants [40,41,[46][47][48][49] (Table 1).…”

“…While all ferric hxHbs bind the distal histidine with equilibrium constants  10 [52], the strength of coordination in the ferrous state is variable ( Figure 4E, Table 2) [48]. Ferrous Ngbs, Cgbs, and SynHb are very tightly coordinated compared to the plant nsHbs, drosophila Hb (DrosHb), and mollusk nerve Hb (Mollusk nHb).…”

“…However, rates of oxygen dissociation from hxHbs are generally too slow for oxygen transport, with the exception of the mollusk nHb, DrosHb, and the Class 2 nsHbs [4,24,41,42,48]. Of the hxHbs for which autooxidation has been measured, rates are generally much faster than the pentacoordinate Hbs [46].…”