Allosteric Regulation and Temperature Dependence of Oxygen Binding in Human Neuroglobin and Cytoglobin

Abstract

Two new globin proteins have recently been discovered in vertebrates, neuroglobin in neurons and cytoglobin in all tissues, both showing heme hexacoordination by the distal His(E7) in the absence of gaseous ligands. In analogy to hemoglobin and myoglobin, neuroglobin and cytoglobin are supposedly involved in O 2 storage and delivery, although their physiological role remains to be solved. Here we report O 2 equilibria of recombinant human neuroglobin (NGB) and cytoglobin (CYGB) measured under close to physiological conditions and at varying temperature and pH ranges. NGB shows both alkaline and acid Bohr effects (pH-dependent O 2 affinity) and temperature-dependent enthalpy of oxygenation. O 2 and CO binding equilibrium studies on neuroglobin mutants strongly suggest that the bound O 2 is stabilized by interactions with His(E7) and that this residue functions as a major Bohr group in the presence of Lys(E10). As shown by the titration of free thiols with 4,4 -dithiodipyridine and by mass spectrometry, this mechanism of modulating O 2 affinity is independent of formation of an internal disulfide bond under the experimental conditions used, which stabilize thiols in the reduced form. In CYGB, O 2 binding is cooperative, consistent with its proposed dimeric structure. Similar to myoglobin but in contrast to NGB, O 2 binding to CYGB is pH-independent and exothermic throughout the temperature range investigated. Our data support the hypothesis that CYGB may be involved in O 2 -requiring metabolic processes. In contrast, the lower O 2 affinity in NGB does not appear compatible with a physiological role involving mitochondrial O 2 supply at the low O 2 tensions found within neurons.

Functional Properties of Neuroglobin and Cytoglobin. Insights into the Ancestral Physiological Roles of Globins

“…The lower P 50 value that was originally recorded for human Ngb was presumably due to in vitro formation of an internal disulfide bridge in the protein (20), which increases the affinity for O 2 (see below). We have also shown that, in contrast to Mb, the O 2 affinity of Ngb is markedly sensitive to changes in pH, and that, depending on the temperature range, both normal and reverse Bohr effects are present (indicating an increase or a decrease in the O 2 affinity, respectively, with a pH increase) (19). Such allosteric properties are uncommon in monomeric proteins and reflect the ability of Ngb to undergo conformational changes that affect heme reactivity.…”

“…However, based on an extensive investigation of O 2 binding equilibria, we have recently shown that the O 2 affinity of human Ngb under physiological conditions of pH and temperature is much lower (P 50 = 7.5 torr) (19), whereby only a small fraction of Ngb in nervous tissues would be saturated with O 2 under normal conditions. The affinity of mouse Ngb at physiological temperature and pH may be even lower.…”

“…However, it appears unlikely that Ngb, which has retained a relatively conserved primary structure during evolution, should be able to bind O 2 reversibly only by means of a metabolically-expensive consumption of reducing equivalents. In human Ngb, the single mutation, of the distal His to a Gln residue greatly stabilizes the O 2 complex and increases the O 2 affinity (19), properties that would be advantageous for O 2 delivery within cells. Remarkably, the distal His, a major determinant of the high autoxidation rate, is invariant among Ngbs.…”

“…The Fe 2+ 7O 2 complex of Ngb is unstable in vitro, whereby formation of ferric heme upon air exposure at room temperature occurs within a few minutes (7,19). Ngb-mediated reversible O 2 binding within neurons can only be possible if an enzymatic met-globin reducing system is present, similar to the met-Hb reductase system identified within red blood cells (23) or to that used in in vitro O 2 equilibrium measurements (19,24). However, it appears unlikely that Ngb, which has retained a relatively conserved primary structure during evolution, should be able to bind O 2 reversibly only by means of a metabolically-expensive consumption of reducing equivalents.…”

A neuroglobin-overexpressing transgenic mouse

“…Possible functions of Ngb include those ascribed to other vertebrate globins, which include O 2 transport, storage, facilitated diffusion, sensing and scavenging, and NO scavenging (Suzuki and Imai, 1998;Weber and Vinogradov, 2001). However, some of these seem unlikely because of the low intracellular concentrations at which Ngb occurs (Brunori and Vallone, 2006) and its unfavorable O 2 -binding affinity under physiological conditions (Fago et al, 2004). On the other hand, Ngb could serve as a sensor of local O 2 and NO concentrations, as proposed by Brunori (Brunori et al, 2005).…”