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Department of Biochemistry, Biophysics, and Molecular Biology, Iowa State University, Ames, Iowa 50011
Correspondence: Address reprint requests to Mark S. Hargrove, Tel.: 515-294-2616; Fax: 515-294-0453; E-mail: msh{at}iastate.edu.
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ABSTRACT |
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 TOP ABSTRACT INTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONACKNOWLEDGEMENTSREFERENCES |
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100 µM-1 respectively, indicate that they are not capable of facilitating oxygen transport. |
INTRODUCTION |
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; Royer et al., 2001; Weber and Vinogradov, 2001). Traditional mammalian Hbs that store and transport O2 have dissociation equilibrium constants in the low µM range (Wittenberg, 1966). Because many spectrophotometric measurements are possible at this concentration range, and as oxygen electrodes are sensitive at these concentrations, it is convenient to measure affinity constants using either equilibrium or kinetic methods. For myoglobin and human hemoglobin, equilibrium constants resulting from either procedure are identical (Olson, 1981a,b).
However, equilibrium affinity measurements cannot be assessed for high-affinity (KD << 1 µM) Hbs using traditional oxygen titration experiments because the low protein concentration required to avoid stoichiometric binding precludes simple spectroscopic measurement, and because it is difficult to measure free ligand concentrations accurately in this concentration range (Giardina and Amiconi, 1981). This is not a problem for some high-affinity Hbs, such as soybean leghemoglobin (Lba), that have simple bimolecular reaction schemes allowing unambiguous assignment of equilibrium constants based on measurement of kinetic rate constants. For Hbs with ligand binding reaction schemes that are much more complex or not yet fully understood, kinetic analysis cannot provide equilibrium constants with confidence (Couture et al., 2000; Dewilde et al., 2001; Hvitved et al., 2001; Trent and Hargrove, 2002; Trent et al., 2001a,b).
A good example of this difficulty is the hexacoordinate class of Hbs. Unlike myoglobin and human hemoglobin that have pentacoordinated heme iron in the unliganded or deoxygenated state, these newly discovered Hbs are hexacoordinated, with heme coordination similar to the bis-histidyl ligation in cytochrome b5 (Arredondo-Peter et al., 1997; Duff et al., 1997). Surprisingly, although cytochrome b5 is completely unreactive toward diatomic ligands, hexacoordinate hemoglobins (hxHbs) bind oxygen rapidly. These proteins were first found in higher plants and named nonsymbiotic hemoglobins (nsHbs) to distinguish them from the symbiotic leghemoglobins (Arredondo-Peter et al., 1998). Two hxHbs have recently been discovered in humans: neuroglobin (Burmester et al., 2000; Dewilde et al., 2001; Trent et al., 2001b), present in brain tissue and in the retina (Schmidt et al., 2003), and histoglobin (a.k.a. cytoglobin) (Burmester et al., 2002; Trent and Hargrove, 2002), expressed in many different tissues. HxHbs have also been found in the cyanobacterium Synechocystis (Scott and LeComte, 2000), and in the single celled alga Chlamydomonas (Couture et al., 1999). Initially hxHbs were predicted to have large affinities for oxygen (Arredondo-Peter et al., 1998), but as several of these proteins have now been investigated in more detail, these conclusions are still in question.
In hxHbs, where the kinetic reaction schemes are more complex than a simple bimolecular reaction, it is imperative that equilibrium constants estimated from kinetic values be tested against experiments that directly measure binding under equilibrium conditions. Their potentially high ligand affinities mean that a new method allowing direct measurement of equilibrium constants is required not only to investigate potential physiological roles, but also as an independent evaluation of kinetic reaction schemes. The method presented here allows direct measurement of equilibrium constants for any Hb regardless of its affinity. It is based on the competition for binding between the Hb whose affinity is to be measured and an array of Hbs with known ligand affinities. Theoretical and experimental details of this method are described along with measurement of equilibrium constants for O2 and CO binding to two different hxHbs. We find that Synechocystis hemoglobin (SynHb) and rice nonsymbiotic hemoglobin (riceHb) have similar affinities for oxygen and carbon monoxide despite their differences in kinetic behavior.
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MATERIALS AND METHODS |
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and Kundu and Hargrove (2003). RiceHb and SynHb were expressed and purified as described by Arredondo-Peter et al. (1997) and Trent et al. (2001a) (for riceHb and riceHbH73L), and Hvitved et al. (2001) (for SynHb). The cDNA for Ascaris Hb domain 1 was kindly provided by Dr. Dan Goldberg. This domain was expressed in Escherichia coli BL21(DE3) cells grown in LB media at 37°C, induced with 0.5 mM IPTG at an optical density of 0.6 at 600 nm, and incubated for an additional 15 h post induction. After cell lysis, recombinant Ascaris Hb was purified as described by others (De Baere et al., 1994; Kloek et al., 1993). The rate constants for oxygen and carbon monoxide binding to recombinant Ascaris Hb were measured using methods described by Kundu et al. (2002). Protein concentrations were measured spectrophotometrically.
Experimental conditions for carbon monoxide binding
All CO experiments were carried out in a volume of 1.5 ml in glass cuvettes (Starna model 29/G/10) with a 1-cm light path in a buffer of 0.1 M potassium phosphate, pH 7.0. Unless otherwise specified, 6 µM each of target Hb and scavenger proteins were reduced with 100 µM sodium dithionite, and the resulting spectrum was collected. A quantity of 6 µM CO was introduced to the reaction mixture by the addition of 9 µl of a saturated (1000 µM) CO stock solution. After a 30-min incubation period to ensure that the reactions were at equilibrium (no spectral changes were observed after this period of time), an absorbance spectrum was measured of the 1:1:1 ratio of Hb:scavenger:ligand. Excess CO (an additional 10 µl of the stock solution) was then added to generate the saturated sample. All absorbance spectra were measured with a Cary-50 Bio spectrophotometer from Varian (Mulgrave, Victoria, Australia), and exported as a text file for use in the SpectraSolve software (described below). Individual component spectra were measured for each protein using the same procedure but with single proteins rather than mixtures.
Experimental conditions for oxygen binding
Oxygen binding was measured in a square 1-cm quartz cuvette fitted with a male ground-glass 
-inch top. The fundamentals of the reduction system have been described previously (Hayashi et al., 1973). Our implementation of this system was as follows: 3 ml of 0.1 M potassium phosphate (pH 7.0) containing 500 µM glucose-6-phosphate (G6P) and 20 µM NADP+ (Sigma, St. Louis, MO) was sealed in the cuvette using a white rubber septum (Aldrich, Milwaukee, WI). The cuvette was then gently bubbled for 20 min with nitrogen flowing through an oxygen scrubbing column (Agilent model OT3-4) and venting through a second needle in the stopper. While maintaining positive pressure, the following were added to the cuvette:
2 mM) of N2 purged, ferric scavenger, and unknown Hbs (final concentration of 6 µM of each).
After addition of these components, the flush and vent needles were removed and the stopper was covered with parafilm. Absorbance spectra were collected at 15-min intervals until both Hbs were in the deoxy form, and no further changes occurred. This was typically 30 to 45 min, but reduction times were variable for different hemoglobins. After collecting the final deoxygenated spectrum, air-saturated buffer was added to bring the O2 concentration to 6 µM. This spectrum was allowed to equilibrate for 20 min (or until no further changes in absorbance occurred) before collection of the final spectrum. The cuvette was then opened to air to saturate the Hbs, and a final saturated spectrum was collected. As with the CO experiments, individual component spectra were measured using the same procedure but with single proteins rather than mixtures.
Spectral fitting
Concentrations of individual species in the mixed spectra were measured by fitting to the individual components collected under the same experimental conditions. Spectra were fit using the program SPECTRA-SOLVE (Ames Photonics), using the sequential simplex method to fit spectral shapes. In some cases (specified in the Results section), liganded and ligand-free species for both the scavenger and unknown Hb could be resolved. In other cases, (as in the CO and oxy-species of riceHb compared to Lba and its mutant proteins), the ligand-bound proteins were fit as a single species and differential ligand binding was determined from the ratio of ligand-free hemoglobins. The specific details of each fitting routine are included in the Results section for each experiment. All figures were prepared using Igor Pro (WaveMetrics).
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RESULTS |
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100) that of the unknown Hb. Second, there must be a measurable spectral difference between either the two deoxy proteins or the two ligand-bound proteins. The first requirement is met here by the pentacoordinate Hbs listed in Table 1 along with their equilibrium constants for O2 and CO. These proteins all exhibit simple bimolecular ligand binding, and their equilibrium affinity constants were calculated as the ratio of association and dissociation rate constants. Their dissociation equilibrium constants (KD) range from the nM to the µM levels, making them convenient for competition with unknown Hbs with potentially high ligand affinities. The second requirement is met for hxHbs because their deoxy spectra are always distinct from those of the pentacoordinate scavengers (Fig. 1 A). In many cases, the ligand-bound spectra are also different (Fig. 1 B).
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Quantification of this phenomenon is achieved using the program SpectraSolve, which fits a mixed spectrum to defined component spectra and returns the relative contributions of each. Fig. 2 illustrates this procedure with riceHb competing for CO against Lbawt (A–C) and LbaH61L (D–F). In each case, the dashed line is the observed mixed spectrum, the dotted line is the fitted spectrum, and the solid line (below) is the residual difference between the fitted and observed spectra. Mixed spectra are shown at 0, 6, and 12 µM CO for both scavenger examples, and the relative contribution of each component spectrum is shown in an inset to each graph. As expected, the deoxy species predominate in Fig. 2, A and D, and the CO-bound species are the majority in Fig. 2, C and F. The component ratios in Fig. 2, B and E indicate that LbaH61L can outcompete riceHb for CO, but Lbawt cannot.
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). For convenience, important relationships specific to the Hb competition method will be stated here. The reaction scheme for a scavenger protein (S) competing with a target Hb (H) for a ligand (L) is shown in Reaction 1 (R1):
 | (R1) |
 | (1) |
HT + ST, then L can be neglected and LT = YHLHT + YSLST. With these substitutions into Eq. 1, a general relationship between the fraction of ligand-bound scavenger (YSL), LT, ST, and HT can be written:
 | (2) |
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 | (3) |
7 times less than that of LbaH61L. As a control experiment, this method was used to evaluate the ratio of CO affinity constants for two known Hbs, Mb and Lba (Fig. 3, E–G). In Fig. 3 E, no CO has been introduced and a fit to these spectra indicates the presence of only the deoxygenated species for each protein. In Fig. 3 F, the concentrations of Mb, Lba, and CO are 1:1:1, and the ratio of CO:Lba to CO:Mb is measured to be 9. Using Eq. 3, this yields a ratio of affinity constants for Lba/Mb of 81. The known values of KCO for Lba and Mb are 2000 and 27 µM-1, respectively (Table 1). The resulting expected value for the ratio of equilibrium constants is 74, and is near that predicted from Fig. 3, E–G.
Equilibrium CO affinity in SynHb and riceHb
Ratios of CO-bound scavenger to CO-bound riceHb or SynHb were measured for each of the CO-scavengers listed in Table 1. In all cases, ST = HT = LT. Results can be reported as both the ratio of CO-bound scavenger to CO-bound unknown Hb (SL/HL), or the ratio of deoxy species (H/S). Experimentally, the two ratios were very similar and the numbers were averaged. The data for both riceHb (solid) and SynHb (open squares) in Fig. 4 show ratios near 1 when assayed against LbaH61A, indicating that each has an affinity constant of 14,000 µM-1. The ratios for both proteins against LbaH61L are 2.3. Using this value along with Eq. 3, a ratio of affinity equilibrium constants for LbaH61L and each hxHb is predicted to be 5.3. When the affinity constant of LbaH61L (Table 1) is divided by 5.3, these experiments agree on a value of 14,000 µM-1 for riceHb and SynHb.
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). But as the oxygen association affinity constant approaches 1 µM-1, this method loses its effectiveness. Another method has been removal of all oxygen from an airtight custom-fitted burette containing a Hb, followed by titration with DT while monitoring optical absorbance. This is unpractical for the high-affinity Hbs studied here, and our experience was that more than stoichiometric DT concentrations were required for reduction. This results in a potential for reduced DT remaining in solution, and little confidence in [O2] once it is added. Stoichiometric DT addition resulted in an initial formation of deoxy Hb followed by an oxy-like spectrum, and subsequent Hb oxidation.
In the experiments defining this competition method, deoxy proteins were generated without DT by purging a cuvette containing the ferric forms of these proteins with O2-scrubbed N2 and then reducing the proteins with the ferridoxin/ferridoxin-reductase system (Hayashi et al., 1973). As the ferric form of the protein binds no oxygen, purging at this step allows complete O2 removal before reduction. Using this system, we were able to generate the deoxy species of each scavenger, riceHb, and SynHb without using DT (an example is shown in Fig. 7).
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100 µM-1) based on competition with Ascaris Hb, but we were unable to make a reliable measurement against LbaH61R.
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)), this combination of scavenger and unknown Hb cannot be used in our equilibrium competition experiment. Of the 13 combinations of oxygen scavenger/unknown Hb measured in this work, this is the only one that behaved unreliably. |
DISCUSSION |
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Extensive kinetic analysis of the reaction scheme for riceHb has led to a model for ligand binding and prediction of equilibrium constants based on kinetic rate constants (Trent et al., 2001a). But in the case of SynHb (and other Hbs with complex reaction schemes), confident estimates of ligand affinities have not been proposed (Couture et al., 2000; Hvitved et al., 2001). Presented here, in addition to a description of the equilibrium competition method, are equilibrium constants for O2 and CO binding to these two hxHbs. The following discussion examines equilibrium versus kinetic measurements of affinity in riceHb and the biological implications of the equilibrium constants measured for both hxHbs.
Equilibrium versus kinetic measurements of affinity constants
The reaction scheme for ligand binding to hxHbs involves "open" and "closed" protein conformations in addition to the hexacoordinate and pentacoordinate states (Trent et al., 2001a).
 | (R2) |
) from R2.
 | (4) |
), KCO = 6000 µM-1, KH = 0.38 (Hargrove, 2000), and KC = 10 (Trent et al., 2001a). These corrected affinity constants are reported in Table 2 along with the uncorrected (i.e., k'L/kL) values and the values measured using the equilibrium competition method.
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There are at least three reasons to have confidence in the results of the equilibrium competition method over those of the kinetic method: 1), Competition for CO between Lba and Mb indicate that the equilibrium competition method can accurately measure the relative CO affinity constant for these two proteins. 2), With both SynHb and riceHb, competition with each scavenger is internally consistent. In no case did a scavenger with lower affinity show appreciable ligand binding in competition with the hxHb. Only when the scavenger affinity reached that of Ascaris Hb (for oxygen) or LbaH61A (for CO) were appreciable concentrations of either deoxy hxHb observed. If there was a problem with a particular scavenger protein, or with the method, one might expect spurious results with at least one of the experiments. In the case of LbaH61R and SynHb, reliable data were not collected, but this reaction did not violate the continuity of the results in the same way that would occur if, for example, wild-type Mb were to outcompete SynHb for O2 but Lba could not. 3), R2 is not a complete model for ligand binding, and only accounts for some of the slow phases of ligand binding after rapid mixing. Several hxHbs were examined by Trent et al. (2001a), and riceHb had the smallest degree of slow-phase ligand binding. For this reason, kinetic estimates of affinity constants would seem to have the greatest chance of being accurate with riceHb. Ligand binding to SynHb is so complex that affinity values have still not been assigned by kinetics (thus the lack of these values in Table 2) (Couture et al., 2000; Hvitved et al., 2001). Although riceHb provides the best chance of measuring accurate affinity constants from kinetic methods, the kinetic model is still not complete enough to challenge a direct affinity measurement.
Biological implications
Numerous physiological functions have been proposed for hxHbs including oxygen transport, ligand sensing, scavenging and destruction of ligands such as oxygen or nitric oxide, and alternative respiratory biochemistry (Dordas et al., 2003; Hunt et al., 2002; Trent and Hargrove, 2002; Van Doorslaer et al., 2003). If the role involves oxygen binding, a fundamental question is whether scavenging or transport is the biochemical objective. Transport by facilitated diffusion for purposes of respiration requires: 1), Hb concentration high enough to augment nonfacilitated diffusion. The solubility of pure O2 in water is 1 mM. For a Hb to facilitate diffusion, its concentration must be in this range or higher. 2), The affinity constant and exogenous ligand concentrations must be such that the facilitating Hb is partially saturated across a concentration gradient. 3), The dissociation rate constants must not limit ligand diffusion (Wittenberg, 1966, 1965; Wyman, 1966). For riceHb and SynHb, these parameters are not within ranges that would enable a role in facilitated diffusion. RiceHb concentrations are thought to be very low in plants (Arredondo-Peter et al., 1997; Duff et al., 1997), and high concentrations of SynHb have not been observed in vivo. Furthermore, both proteins have oxygen dissociation rate constants that are very slow. In the case of riceHb, oxygen dissociation is slowed by a hydrogen bond with the distal histidine (His73); in SynHb, the structural factors causing slow dissociation are less clear and involved multiple amino acids (Arredondo-Peter et al., 1997; Couture et al., 2000; Hvitved et al., 2001).
The results presented here indicate that although oxygen affinities are lower than those predicted from kinetic experiments, they are still too large to allow facilitated diffusion to occur under any known physiological conditions. Both proteins possess affinity constants for oxygen that are similar to that of Ascaris perienteric Hb, a protein that does not facilitate oxygen diffusion even under micro aerobic conditions (Wittenberg, 1966; Wittenberg et al., 1974). Our results also indicate that the current kinetic model for ligand binding to hxHbs is not sophisticated enough to accurately predict equilibrium constants from kinetic rate constants. It is possible that the kinetic rate constants for the slow phases described for hxHbs (Dewilde et al., 2001; Trent and Hargrove, 2002; Trent et al., 2001b) are ligand specific, or that the dissociation rate constants have an additional component that is extremely slow which has not yet been accounted for. Whatever the reason is for the lack of agreement between kinetics predictions and equilibrium measurements, it almost certainly arises from the limited kinetic model. A combination of kinetic and equilibrium methods is therefore required to evaluate the biophysical behaviors of these proteins.
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ACKNOWLEDGEMENTS |
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Submitted on November 4, 2002; accepted for publication January 28, 2003.
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