Integrative and Comparative Biology Advance Access originally published online on August 20, 2007
Integrative and Comparative Biology 2007 47(4):656-661; doi:10.1093/icb/icm073
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Negative cooperativity in Root-effect hemoglobins: role of heterogeneity
Institute for Molecular Biophysics, Johannes Gutenberg University, Mainz, Germany
Correspondence: 1E-mail: hdecker{at}uni-mainz.de
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In some animals, the oxygen transport capacity of blood decreases when pH is lowered, yielding oxygen binding curves with Hill-coefficients smaller than unity. This so-called Root effect is observed in several fishes and is important for creating large oxygen partial pressures locally, for example in the swim bladder. While there is general agreement on the physiological advantages of this effect, its molecular basis remains ambiguous. Various studies show that isoforms of hemoglobins usually are present in the hemolymph, when the Root effect is observed. Here, we show that in such a case the mixture of these isoforms can exhibit apparent negative cooperativity, although each component taken separately can be described by the MWC model. In other cases, isolated isoforms exhibit true negative cooperativity. The well established MWC model describes many cooperative phenomena of enzymes and respiratory proteins but is not capable of describing negative cooperativity. In order to model negative cooperativity within a single molecular species a decoupling model might be employed, as pointed out previously. However, simulations show that it is not mandatory to have species with negative cooperativity, in order to obtain the binding curves typically seen for whole blood. These two aspects of the Root effect will be discussed on the basis of data from the literature.
Hemoglobins (Hb) as respiratory proteins are well investigated and understood. The tetrameric Hb binds oxygen cooperatively with high affinity at the respiratory surface (lungs and gills) and with low affinity at the tissues. Its oxygen-binding behavior can be modulated by allosteric effectors such as 2,3-diphosphoglycerate or protons. The latter is known as the Bohr effect. Often an increase of proton concentration lowers the affinity of the blood but a complete saturation at ambient pO2 is still possible and often at very low pH-values an acidic Bohr effect is observed. In contrast to this effect is the Root effect (Root 1931
) observed in the Hb of many fishes. The Root effect is characterized by a very strong decrease in affinity to the extent that the blood cannot be oxygenated totally at ambient partial pressure of oxygen. Furthermore, positive cooperativity is reduced and can even switch to apparent negative cooperativity. We use the term apparent negative cooperativity, when we merely refer to a phenomenon which is defined by a Hill-coefficient which is smaller than unity, without further distinction with respect to the underlying mechanism. In addition, the acidic Bohr-effect usually is missing (Brittain 2005). Thus, at low pH the blood remains partially deoxygenated. The Root effect is essential for generating locally hyperoxic oxygen partial pressures, in order to fill the swim bladder of fishes with gaeous oxygen and to ensure a sufficient oxygen supply to the retina in the eyes (Feuerlein and Weber 1996; Pelster 2001, 2004; Pelster and Decker 2004; Berenbrink et al. 2005; Brittain 2005). The Root effect occurs mostly in the blood of teleost fishes and has also been reported for the blood of elasmobranches and amphibians. While general agreement on the physiological advantages of this effect has been reached, its molecular basis is still ambiguous.
The molecular mechanism of the Root effect is still currently debated and began with the assumption of Max Perutz and others (Perutz and Brunori 1982) that the presence of the amino acid serine in position ßF9 was crucial for establishing a Root-effect Hb. Meanwhile, it seems that several other amino acids are also involved in the Root effect and even the function of position ßF9 is debated (Brittain 2005). Several approaches were reported for explaining the Root effect on the molecular level (Perutz and Brunori 1982; Ito et al. 1995; Mylvaganam et al. 1996; Perutz 1996; Tame et al. 1996; Mazzarella et al. 1999, 2006a, 2006b; Tamburrini et al. 2001; Tsuneshige et al. 2002; Verde et al. 2002; Yokoyama et al. 2004). While only one proton is released per tetramer in human Hb at pH 7.4 in a T-to-R transition provided by histidines in absence of chloride (Perutz and Mathews 1966), this number is raised to about four to eight delivered by additional amino acids involving an Asp-cluster in the Antarctic fish Pagothenia bernacchii (Camardella et al. 1992). The amino acids responsible were also proposed by modeling the structure on the basis of the respective sequences and the structure of human Hb (Verde et al. 2003).
Amino acids additional to those responsible for the Bohr effect are required for an explanation of the drastic reduction in the Hill-coefficient to about unity and in some cases they seem to be arranged in clusters (Mazzarella et al. 1999, 2006b; Tamburrini et al. 2001). In the case of the cathodic Hb of the Antarctic fish Trematomus newnesi the cluster of three aspartates (Asp95
, Asp101ß, Asp99ß) is considered to be the minimum structural requirement for establishing the Root effect (Mazzarella et al. 2006a).
In all these attempts to gain information about the molecular origin of the Root effect, the modulation by protons is regarded to be localized merely on one Hb isoform and is thought to have originated from a strong proton-dependent change in free energies of the low affinity T-state relative to the high affinity R-state. The T
R transition seems to be inhibited in Root Hbs. However, there does not seem to be any unique mechanism in terms of the shift of allosteric equilibria induced by protonation of the relevant residues. The protonation of positive-charge clusters in the Hb of Leiostomus seems to destabilize the R-state of the Hb (Mylvaganam et al. 1996). Several authors hold an over-stabilization of the T-state to be responsible for the Root effect. Furthermore, there seems to be no general pattern with respect to the localization of these charge clusters: do they lead to stabilization of a particular conformation on the basis of a single subunit or are they located between subunits, connecting their conformational transitions? Indications for the latter concept was given by Mazzarella et al. (2006a, 2006b) and Tsuneshige et al. (2002). Taking all this information into consideration, it appears quite likely that different types of amino-acid clusters may cause the Root effect, and this suggests that the Root effect has been "invented" more than once during evolution (Berenbrink et al. 2005).
Another question to be answered is: What conformations are involved in this regulatory process? Thus, a suitable model for the allosteric effects has to be found. The approaches that can be used to address this question depend on whether this effect is based on individual Hb molecules or on an ensemble of them. Most available data are based on oxygen-binding measurements of whole blood and not on isolated isoforms. Unlike the majority of mammals, which produce a single major Hb component, many fish species have multiple Hb components that show considerable differences in amino-acid sequence These isoforms can be grouped into cathodic and anodic Hbs according to their behavior in gel electrophoresis. The cathodic Hb typically has a pI>8, exhibits no Root effect and has a high affinity for oxygen. In contrast, anodic Hb typically has a pI<8, a low affinity, and both cooperativity and affinity are strongly pH dependent, yielding a large Bohr effect and/or Root effect (Binotti et al. 1971; Pelster and Weber 1991; di Prisco and Tamburrini 1992; Fago et al. 1995, 1997; Verde et al. 2003). The ratio between the concentrations of these two types is species-dependent. The occurrence of Hb isoforms does not necessarily lead to the establishment of a Root effect and these Hb components do not always show functional differences. For instance, the two major Hbs from carp are not functionally distinguishable from each other (Tan et al. 1972), whereas the Hb systems of trout, eel, and moray have two types of Hb that differ markedly in their structural and functional properties. Furthermore, the correlation between pI and establishment of the Root effect is not strict, since examples are reported in which the cathodic Hb displays the Root effect, whereas the two anodic components do not (Trematomus newnesi) (d’Avino et al. 1994; d’Avino and De Luca 2000) or in which one single anodic Hb is found that is devoid of the Root effect [Gymnodraco acuticeps, Aethotaxis mytopterix, (Tamburrini et al., 1992)].
In any case, at least these isoforms which strongly response to a shift in pH has to be present, in order to exhibit the Root effect in whole blood. This strong response might be considered merely as a very pronounced Bohr effect and be basically explained within the framework of the MWC-model. There has been serious objections to the proposal that the Root effect is just a very strong shift from R-state Hb to T-state Hb (Brittain 2005). Firstly, an increasing number of experiments indicate that, in general for Hb, more than two conformations are needed to explain the modulation of oxygen-binding properties by various effectors, including protons (Connelly et al. 1986; Weber et al. 2000; Yonetani et al. 2002; Ackers and Holt 2006). Furthermore, it was proposed that Root-effect Hbs show additional features compared to the "normal" Bohr effect: Root-effect Hbs completely lose their cooperativity, in contrast to Bohr-effect Hbs and they do not show any acidic Bohr effect (Brittain 2005).
In the following discussion we want to explore the different possibilities, whereby the observed pH-induced loss of binding capacity in blood exhibiting the Root effect can be explained, based on models for cooperativity, and to shed light on the question whether or not the Root effect can be regarded as an enhanced Bohr effect.
The experimental data for trout hemoglobin HbI and HbIV obtained by Binotti et al. (1971) serve as the basis for our simulations. The oxygen-binding behavior of isoform HbIV is strongly pH dependent: between pH 8.0 and pH 7.1 the log p50 increases from 1.2 to 2.3 with a relative stable Hill-coefficient of about 2. A further decrease of pH leads to further increase in p50 and concomitant decrease in the Hill-coefficient, until at pH 6.12 finally a curve with negative cooperativity is observed. In contrast, the binding characteristics of trout hemoglobin HbI are unaffected by changes in pH. We interpolated the data of these purified two isoforms (Fig. 1A and B), in order to simulate a mixture, as typically found in the blood, with relative amounts of HbIV/HbI
1/3 (Brunori 1975). The interpolations are based on a data analysis either with the MWC-model or the Hill-equation (see also figure legend). The saturation degree 
for the MWC-model is given by the following equation
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KR, KT are the binding constants for the R-state and the T-state, L the allosteric equillibrium constant in absence of ligands and x the ligand activity. Since the binding curves do not extend very far to the low and high saturation level, the parameters obtained from the fit are not well defined and therefore are not given. In order to simulate a mixture, the saturation degrees for the two isoforms (IV and I) were added according to = (1/4)IV + (3/4)I.
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The results of these simulations as shown as binding curves (Fig. 2) and as Hill- plots (Fig. 3). The curves at pH 7.1 or lower show strong negative cooperativity. The slope of the mixture at pH 7.1 exhibits a Hill-coefficient of about 0.6 in the flattest region. For comparison: the Hill-coefficient of the isoform HbIV at this pH is 2! This example demonstrates that when negative cooperativity is found the simplest explanation is to assume two different isoforms. Thus, in order to understand the underlying mechanism it is mandatory to clarify whether or not one is dealing with isoforms with different oxygen-binding characteristics. The importance of identifying and analyzing the isoforms individually has been recognized. What seems to be less clear is that the extent of (apparent) negative cooperativity in whole blood and in the isolated isoforms can differ significantly and it seems to be difficult to find a physiological correlation between the two characteristics. Furthermore, when comparing whole blood and isolated isoforms, one has to make sure that in both cases the same composition, with respect to effectors such as organic phosphates, are present. There are also examples in the literature in which isolated isoforms exhibit negative cooperativity on their own, for example, Trout IV (Binotti et al. 1971
), bluefish (Bonaventura et al. 2005), tuna (Yokoyama et al. 2004), and anodic eel (Fago et al. 1995). Here, clearly the MWC-model is not suitable to describe oxygen-binding behavior. Since Hbs consist of two types of subunits the simplest idea is to assume that lowering the pH leads to a decoupling of the subunits, yielding in a functional heterogeneity within the tetrameric Hb (Fago et al. 1993; Coletta et al. 1996; Ascenzi et al. 2005)
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The data for trout HbIV presented by (Binotti et al. 1971
) show negative cooperativity at pH 6.12, but this is based on only two data points. In a later study data on oxygen binding, data were obtained at 14°C (compared to 20°C in the earlier study), showing that the negative cooperativity at pH 6.1 is well established, whereas at pH 6.7 trout HbIV is still binding oxygen with positive cooperativity (Brunori et al. 1978). The binding curve of HbIV at pH 6.1 can well be described by a model in which two subunits have an affinity of about 0.004 Torr–1 (p50 = 250 Torr) and the other two subunits an even lower affinity of about 0.0004 Torr–1 (p50 = 2500 Torr; fit and data not shown). However, inspection of Fig. 2A and B reveals that the differences in the binding behavior of a mixture of HbI and HbIV at pH 6.1 (HbIV negative cooperativity) and 6.5 (HbIV positive cooperativity) are not very large. Thus, one might wonder what additional physiological relevance these uncoupled Hb might have.
Is the Root effect an enhanced shift between T-states and R-states, or not? In cases in which the observed Root effect in whole blood is mainly produced by a mixture of isoforms, which differentially respond to changes in pH values, but none of them individually displaying negative cooperativity, the answer to this question would be "yes" in the sense that no additional features, compared to those found in other Hb systems, have to be introduced. The common scheme is that there are a number of cooperatively-acting conformations whose relative amounts are regulated by various effectors. However, this conformational system might be more complex than a simple MWC-model, as indicated by an increasing number of experiments on Hbs not showing any Root effect (Henry et al. 2002; Imai et al. 2002; Yonetani et al. 2002).
In those cases in which clearly isolated isoforms exhibit negative cooperativity, an additional feature needs to be introduced. In order to describe the function of these isoforms, one has to assume a functional decoupling of the tetramer into its heterogeneous components (Brunori et al. 1978; Olson et al. 1987; Ackers and Holt 2006). The following model could apply to the change in cooperative behavior as found in trout HbIV: the tetramer can exist in two states. In one state, the whole tetramer is functionally coupled and forms an allosteric unit, as described in the MWC-model. In the other state, the subunits are completely decoupled and the functional properties resemble those of a heterogeneous mixture of - and ß-chains. These two states are in equilibrium with each other. Furthermore, the equilibrium between these two states is pH-dependent: at low pH, the uncoupled state prevails but it vanishes at high pH. This decoupling is a new feature that was not necessary for the description of the regulation of other Hbs. Thus, a Root effect can be established in whole blood, (1) by a single Hb isoform that is capable of decoupling or (2) if at least two different Hbs are present that differ in their binding properties. These two situations differ in the mechanistic approach used to generate the Root-effect in whole blood. The two cases should be treated separately, although they share a common feature in comparison to the Bohr effect: the response to reduced pH-values is stronger and it requires more amino acids, which can be protonated/deprotonated in the relevant pH-range. Also a mixture of these two cases can be found, when hemoglobin isoforms with negative cooperativity are present, but additionally other isoforms can be found as reported for some Artic and Antarctic fishes (Tamburrini et al. 1997; Verde et al. 2002). Ongoing work is addressing the physiological role of the Root-effect, but one may wonder whether the anodic (usually negatively cooperative) Hbs are responsible for generating hyperbaric oxygen tensions in very localized regions of the organism, such as the fish swim bladder or the retina. Possibly, in contrast to the Bohr effect, the Root effect rather operates locally than at the level of the whole organism.
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This paper summarizes one of the 22 symposia that constituted the "First International Congress of Respiratory Biology" held August 14–16, 2006, in Bonn, Germany.
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