©1995 by The American Society for Biochemistry and Molecular Biology, Inc.
A Spectroelectrochemical Method for Differentiation of Steric and Electronic Effects in Hemoglobins and Myoglobins (*)

Kevin M. Faulkner (1), Celia Bonaventura (2)(§), Alvin L. Crumbliss (1)(§)

From the (1) Department of Chemistry, Duke University, Durham, North Carolina 27708-0346 and the (2) School of the Environment Marine Laboratory, Duke University, Beaufort, North Carolina 28516-9721

ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
FOOTNOTES
ACKNOWLEDGEMENTS
REFERENCES

ABSTRACT

Spectroelectrochemical techniques are described which enable us to compare anion effects on redox curves of structurally distinct hemoglobins with oxygenation curves obtained under equivalent conditions. Nernst plots for tetrameric vertebrate Hbs show evidence of cooperativity, with the T state conformation more resistant to oxidation than the R state. Anions shift the conformation toward the T state and decrease the ease of oxidation, with variations in anion sensitivity similar to those observed in oxygen equilibria. Oxygen binding, unlike electron exchange, is known to be subject to steric constraints that vary considerably in natural and engineered hemoglobins that have differences in the distal residues of the heme pocket. Since oxidation curves are not subject to steric hindrance, anion-induced differences between the oxidation and oxygenation curves can be indicative of anion-induced alterations in the stereochemistry of the heme pocket that alters the ease of ligand entry or exit. Addition of inositol hexaphosphate to solutions of Hb A in 0.2 M nitrate generates such differences: the ease of electron abstraction from deoxy (T state) Hb A is unaffected, while, as previously reported, the oxygenation of deoxy (T state) Hb A is greatly hindered. The difference between inositol hexaphosphate effects on initial stages of oxidation and oxygenation indicates that the explanation for ``multiple T states'' in oxygen binding lies in the ability of the polyanion to greatly increase steric hindrance to ligand entry, without appreciable changes in the electronic features of the heme environment.


INTRODUCTION

Hemoglobins of diverse species have structural features that allow organisms with widely differing physiological demands to exist in a wide range of environments. The basis for this functional flexibility, which has intrigued researchers over the years, lies in the ability of strategic amino acid residues to alter the protein's intrinsic oxygen affinity and to respond to metabolites that exert allosteric effects. These direct and indirect control mechanisms modulate oxygen affinity efficiently and elegantly.

Although there is a large body of literature that describes the direct and indirect mechanisms involved in controlling hemoglobin function, there are still unanswered questions with regard to how globin structure controls the electron donating or withdrawing potential of the active metal sites. A linkage between ease of iron(II) heme oxidation and oxygen transport is present at a fundamental level, since loss of electrons results in loss of oxygen binding capability. Loss of electrons occurs spontaneously in vivo and the oxygen-binding state is restored by electron-donating systems (Kiese, 1974). Activated oxygen species can be formed in the process, with potentially adverse physiological effects (Winterbourn, 1985; Misra and Fridovich, 1972; Alayash et al., 1992).

The intrinsic redox potential of heme proteins is typically determined for samples in the absence of oxygen. It is important to differentiate between studies of redox potential and studies of autooxidation, which concerns the rate of oxidation of hemoglobin solutions in the presence of oxygen. Autooxidation mechanisms are the subject of continuing debate (Brantley et al., 1993; Shikama, 1984) since the process has a complex dependence on subunit dissociation, oxygen partial pressure, pH, and anion concentration (Rifkind, 1974). Early quantitative studies of the oxidation-reduction equilibria of respiratory heme proteins (in the absence of oxygen) were carried out by Taylor and Morgan(1942) who studied the equilibrium between ferrous and ferric myoglobin. As reviewed elsewhere (Antonini and Brunori, 1971), the oxidation-reduction equilibrium of hemoglobins has been the subject of many earlier investigations, with inconsistencies as to the shape of the oxidation-reduction curves. These inconsistencies have been attributed to the use of oxidizing or reducing agents that interact with the protein. Early workers in the field, recognizing that protein-dye interactions could create artifacts, introduced a ``method of mixtures'' to eliminate the use of reducing dyes. The method involved mixing varied proportions of deoxygenated ferrous and ferric hemoglobin and reading the potential after a period of equilibration in the presence of an electron-transfer mediator. This method has not been used in recent years and was complicated in early studies by incomplete removal of ferri-ferrocyanide from the ferric hemoglobin (Antonini et al., 1964; Desbois and Banerjee, 1975).

In previous studies of the redox potential of hemoglobin, the general conclusion was drawn that the oxidation potential is sensitive to protein conformation, with a greater ease of oxidation for hemoglobins that show high oxygen affinity. Hemoglobin digested with carboxypeptidase A, used as a model system representing the high affinity conformation of hemoglobin, was shown to lose electrons much more readily than the parent protein (Antonini and Brunori, 1971). The results presented herein extend these studies and help clarify the dependence of the redox equilibrium on the quaternary conformational equilibrium of hemoglobin.

The conformational transition from the T state, of low oxygen affinity, to the R state, of high oxygen affinity, is reflected by slopes of greater than one in Hill plots of oxygen binding. This transition also results in cooperativity in the oxidation process and is reflected by slopes of greater than one in Nernst plots (see ``Experimental Procedures''). Chain heterogeneity is more apparent in the oxidation curves than in the oxygenation curves, resulting in slopes of the Nernst plots of the oxidation process that are typically less than in the Hill plots of the oxygenation process (Antonini and Brunori, 1971). Comparison of both hemoglobin oxidation-reduction and oxygenation-deoxygenation requires consideration of a 6-fold equilibrium scheme as illustrated below (). This is based on the Monod, Wyman and Changeaux two-state model (Monod et al., 1965) and provides for an R T equilibrium shift associated with both the oxygenation and redox processes.

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SCHEME I

In light of the inconsistencies in the experimental design in past redox studies of Hb (Antonini and Brunori, 1971; Antonini et al., 1964; Desbois and Banerjee, 1975; Brunori et al., 1964;Song and Dong, 1988; Dong et al., 1989), and the recent synthesis of a synthetic redox protein which exhibits anti-cooperativity (Robertson et al., 1994), it is timely that the Hb redox system be thoroughly re-examined. Our spectroelectrochemical approach allows us to explore the redox behavior of Hb with higher resolution and reproducibility than was previously possible. Moreover, since the mediator used in our study is not an effector of hemoglobin function, this approach allows us to make quantitative appraisals of how allosteric effectors influence the redox potential. In this work we have compared changes in the redox potential to changes in oxygen affinity, making use of hemoglobin variants, isolated chains, and a number of different experimental conditions. Carboxypeptidase-digested hemoglobin (HbCPA) was used as a model for R state hemoglobin (Antonini and Brunori, 1971) and human hemoglobin A (HbA) in the presence of excess inositol hexaphosphate (IHP)() was used as a model for T state stabilized hemoglobin. Other hemoglobins studied, which exist in R/T state equilibrium in the absence of added effectors, include trout hemoglobin (HbTroutI), HbA, and bovine hemoglobin (HbBv), in order of increasing shift of the R/T equilibrium toward the T state. We focus in this report on heterotropic allosteric effects of anions on the oxidation and oxygenation processes and show that electronic and steric contributions to hemoglobin functionality can be distinguished by use of this experimental approach.


EXPERIMENTAL PROCEDURES

Materials

Ru(NH)Cl (Strem Chemical Co., >99%), NaNO (Fisher Scientific, >99%), KCl (Fisher Scientific, >99%), MOPS (Sigma, >99%), IHP (myo-inositol hexakis[dihydrogen phosphate] dipotassium salt) (Sigma, >95%), myoglobin (lyophilized solid from horse skeletal muscle, Sigma, >96%), and platinum (52 mesh gauze, Fisher Scientific, 99.95%) were used as obtained. Doubly distilled water was used in all experiments.

Samples of chromatographically purified human hemoglobin A (HbA), carboxypeptidase-digested hemoglobin (HbCPA), and chains from fetal Hb were prepared by standard methods, including lysis of red cells and separation of Hb from lipids by ammonium sulfate precipitation (Antonini and Brunori, 1971). Trout I Hb was a generous gift from Professor Maurizio Brunori of the University of Rome. Samples were stripped of organic phosphates prior to chromatographic purification. Sample concentration and compositions were determined spectrophotometrically as described in the literature (Greenwald, 1985). The amounts of oxidized Hb (metHb), oxygenated Hb (oxyHb), and hemichrome were determined by spectral analysis. Samples that contained measurable amounts of hemichrome were discarded. The final concentration of the stock Hb solution was typically 1-2 mM in heme units. Hb was stored after preparation in liquid nitrogen or at +4 °C until further use.

Methods

Preparation of Samples

The electrochemical mediator, Ru(NH)Cl, was dissolved to give a concentration of 3-5 mM in the desired buffer/electrolyte solution. MOPS buffer was used due to its non-complexing nature and stability. Trizma (Tris base) buffers are unsatisfactory in that the system is slow to come to equilibrium and they cause an increase the rate of hydrolysis of Ru(NH)Cl. The mediator/buffer solutions were stored under argon at 4 °C. For a given experiment, a desired amount of mediator solution was added quantitatively to about 500-700 µl of the proper buffer/electrolyte in a 5-ml flask and connected to a vacuum line for repeated pump-purge with argon, followed by the addition of Hb and additional pump-purging with gentle swirling to minimize bubbling. Final concentrations were typically 0.35-1.0 mM in Ru(NH)Cl, and 0.050-0.20 mM in heme. All data were collected in MOPS buffered solution at pH 7.1. All met hemoglobins were in the aquamet form with negligible amounts of the hydroxymet form.

Stock IHP solutions were prepared by dissolving a weighed amount of the solid into the desired buffer-electrolyte solution, followed by addition of 1 N NaOH and dilution to the desired volume and pH. For experiments where IHP or other phosphates were required, additions were made to the mediator/buffer solution from the concentrated stock solution prior to the addition of stock Hb solution. The IHP concentration was 10-fold over the Hb (tetramer) unless otherwise noted.

Oxygen Binding and Spectroelectrochemistry

Oxygen equilibria measurements were performed tonometrically, with the spectrophotometric method of Riggs and Walbach(1956).

Spectroelectrochemical experiments were carried out in an anaerobic optically transparent thin layer electrochemical cell (OTTLE) made of a 1 2-cm piece of 52 mesh platinum gauze placed between the inside wall of a 1-cm path length cuvette and a piece of silica glass held in place with a Tygon spacer. This arrangement results in an optical path length of about 0.055 cm as calculated by the absorbance of Hb at 555 nm, = 12,500 M cm (Antonini and Brunori, 1971). An air-tight seal was formed at the top of the cell with a rubber septum (Aldrich), through which protruded a connecting platinum wire to the working platinum gauze electrode and a Pasteur pipette salt bridge plugged at the end with agar for the Ag/AgCl reference (BioAnalytical Systems, Inc.) and platinum auxiliary (when used) electrodes. The salt bridges were filled with supporting electrolyte purged with Ar, which was passed through a Chromopack oxygen scavenger. The OTTLE was purged with argon for 15 min prior to injecting protein samples. Hb remains in the deoxyHb state at open circuit for at least 4 h without mediator, and 6-8 h with mediator at closed circuit.

In a typical experiment about 500 µl of Hb/mediator solution were transferred via gas-tight syringe to the argon-purged OTTLE. The OTTLE cell was placed in a temperature-controlled cell holder in the sample compartment of a CARY 2300 UV-Vis-NIR spectrophotometer. Potential control of the three-electrode system was achieved by using a PAR model 175 potentiostat in conjunction with a PAR model 173 programmer for cyclic voltammetry studies of Ru(NH)Cl. All potentials are quoted relative to the Ag/AgCl electrode. The E of the Ru(NH) is -150 mV at a platinum button electrode and in the OTTLE cell containing 0.05 M MOPS, 0.20 M KCl, pH 7.15.

Spectroelectrochemistry was carried out at a single wavelength, typically one of the protein Soret bands (430 nm, = 1.33 10M cm for deoxyHb and 406 nm, = 1.62 10M cm for ferric(met) Hb) (Antonini and Brunori). The potential of the working electrode was held at +200 mV to oxidize all Hb to metHb. The absorbance was then set at zero. Next, the potential was jumped to some reducing potential where the amount of reduced material was about 5% (typically +20 to 0.0 mV), and held until the absorbance ceased to change (typically 5-15 min). Potential jumps went from oxidizing to reducing potentials in 10-20 mV increments. The final potential jump was made from a potential where about 95% of the material was reduced (-160 to -200 mV), to -450 mV. At this extreme negative potential all material is in the reduced state. The absorbance at -450 mV is taken as the total absorbance (A) of the Hb in the deoxyHb state. The spectroelectrochemical process can also be carried out in the oxidative direction. In this case the initial potential was typically about -450 mV. Next, the potential was taken to about -200 mV and the procedure continued in reverse from that described for the reductive direction. In our conditions and with Ru(NH)Cl as a mediator, the redox process is chemically reversible for Hb A in the oxidative and reductive directions. This was apparent by the relative rates of oxidation and reduction of Hb in a given experiment and by the superposition of Nernst plots obtained by oxidative and reductive potential changes. The rate of oxidation of Hb was slower than the rate of reduction, but still rapid on the time scale of an experiment. Total reduction of metHb in a single step takes about 10 min, while total oxidation of deoxyHb in a single step takes about 20 min.

Data Analysis

The absorbances of the Hb Soret band at various potential values were converted to a ratio of [oxidized form]/[reduced form] by using Equation 1, where A is the absorbance at 430 nm when the potential of the working electrode was -450 mV and Hb was totally reduced, A is the absorbance at 430 when the potential of the working electrode was +200 mV and Hb was totally oxidized, and A is the absorbance at 430 nm at each potential E. Since the absorbance of the

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totally oxidized hemoglobin was set to zero (A = 0), Equation 1 simplifies to Equation 2.

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The log of this ratio plotted as a function of potential constitutes a Nernst plot as expressed in Equation 3, where E is the potential of the working Pt gauze electrode controlled by the potentiostat, E is

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the reduction potential (log[oxid]/[red] = 0), n is the slope, and 58.1 is the value of RT/F at 20 °C. For a complex multicentered redox system, the formal redox potential, E`, is difficult to define. Therefore, we define the E value as the value of E at half-oxidation, as is done for the case of non-ideal Nernstian behavior (Kristensen, 1991). For a non-interactive system, the Nernst plot will be linear, and the slope n is indicative of the number of electrons transferred. Thus n would be expected to be unity for Mb and 2 for DCPIP. The complex nature of a multicentered redox system such as described here precludes the determination of electron stoichiometry. For an interactive multicentered redox system the Nernst plot will not be linear and the slope n, which is not constant, will be indicative of that interaction and therefore will not correspond to the number of electrons transferred (Kristensen et al., 1991). Thus the n parameter, as obtained from the slope of Equation 3, may be interpreted in a similar manner to the n parameter one obtains as the slope of a Hill plot of O binding data for systems with interactive active sites, where n values greater than 1 are an indication of cooperative interactions.

The spectroelectrochemical data were plotted according to Equation 3 using a curve-fitting program (``Prostat'' Ward and Reeves, IBM Version) which allows a first derivative plot of the fitted curve to be taken. A sample Nernst and corresponding first derivative plot for human hemoglobin A is shown in Fig. 1. Due to the precision in our results, we are able to define two n values. The n value is the slope of the Nernst plot at E. Often, the maximum value of the slope, n, was found at potentials greater than the E value, thus giving rise to a parameter we define as En (En - En. The n, n, and En parameters are illustrated in Fig. 1. En is dependent on experimental conditions and is used to describe the asymmetry of the Nernst plot. The E and En values are related to the and values described in the two-state model for O binding (Rubin and Changeux, 1966).


Figure 1: The lower line is a Nernst plot of Equation 3 for human hemoglobin A (Hb A). The upper line is a plot of the changing slope of the Nernst plot multiplied by 58.1. These data serve to illustrate the parameters E, n, n and En. Decreasing E values are indicative of increasing ease of oxidation.



Experiments reported herein were reproduced at least twice, and the reproducibility of the various spectroelectrochemical parameters at different buffer and background electrolyte conditions is as follows: in 0.05 M MOPS, E = ±3 mV, n = ±0.10 units; in 0.05 M MOPS, 0.20 M KCl, MOPS/KCl salt-bridge, E = ±2 mV, n = ±0.10 units; and in 0.05 M MOPS, 0.20 M NaNO, saturated NaNO in salt-bridge, E = ±1 mV, n = ±0.05 units.


RESULTS

Spectroelectrochemical Method

Our spectroelectrochemical technique was tested on three thoroughly studied redox systems: DCPIP, methylene blue, and horse myoglobin (hMb). For hMb we confirmed by coulometry that the total number of electrons transferred (n) is 1, and literature reports of coulometric experiments on DCPIP and methylene blue establish these to be two electron redox processes (n = 2) (Fultz and Durst, 1982). Our spectroelectrochemical results for DCPIP, methylene blue, and hMb are compared with literature data in , with excellent agreement. Plots of log[ox]/[red] versusE/58.1 were linear with slopes of 1 and 2 for DCPIP and methylene blue, respectively. Ideal Nernstian behavior as defined by Heineman and co-workers (Kristensen, et al., 1991) was observed with our spectroelectrochemical method for these simple redox systems. We conclude that the spectroelectrochemical method employed in this work is a valid procedure for obtaining parameters related to the electron-transfer properties of light-absorbing molecules.

Hexammineruthenium(III/II) (Ru(NH)), the cationic mediator used in our electrochemical studies, was selected for use because its redox potential is in the correct range and because hemoglobin function is relatively insensitive to cations. The mediator's positive charge precludes interaction with the anion-binding sites of hemoglobin. Hexammineruthenium(III/II) could not be used in oxygen binding studies (where the solution potential is not controlled) because its ability to exchange electrons with the heme iron results in partial heme oxidation, decreased oxygen affinity, and decreased cooperativity. Data presented in demonstrate that the mediator concentration can be varied from 0.2 to 2.2 mM over a heme concentration range from 0.07 to 0.4 mM without significant alteration of the observed redox potential. The resultant Cl mediator counter-ion concentration range did not influence the protein redox or oxygen-binding properties. Experiments at variable Ru(NH) mediator concentrations were also carried out at 0.2 M Cl and NO background electrolyte concentrations () with no observed mediator effect on the results.

MOPS buffers, with varied levels of nitrate as the background electrolyte, were found to work well in electrochemical experiments with Ru(NH)Cl as the redox mediator. With proteins in 0.2 M MOPS, fairly rapid electron exchange is possible in electrochemical studies without addition of nitrate or other background electrolytes. To investigate anion effects it is often useful to use lower buffer concentrations. Experiments can be done with good reproducibility in 0.05 M MOPS, although at this concentration electron exchange rates are significantly slower. The electron exchange occurs faster in 0.05 M MOPS if a background electrolyte is added.

Comparison of Nernst and Hill Plots

Our experimental methods were designed to allow for a detailed comparison between Nernst plots for the oxidation process (such as shown in Fig. 1 ) and Hill plots for the oxygenation process for selected test proteins under comparable experimental conditions. I lists the redox parameters for the different proteins investigated under varied conditions. documents the parameters of the oxygenation processes under comparable experimental conditions.

The E for hMb in 0.05 M MOPS and 0.2 M NaNO was determined to be -160 ± 3 mV, in reasonable agreement with values reported for hMb in other buffer systems (Ellis, 1986). Non-cooperative Nernst plots were obtained for hMb and for chains isolated from human fetal Hb () in reasonable agreement with previous reports (Abraham and Taylor, 1975). For Hb A and HbCPA, the redox potentials are in accord with previous reports, within the expected variations due to differences in experimental conditions (Brunori et al., 1964; Antonini et al., 1964; Ye and Baldwin, 1988).

Fig. 2 compares Nernst and Hill plots for the oxidation and oxygenation of hMb and Hb A under varied conditions, with Ru(NH)Cl as a mediator (see ``Experimental Procedures''). The Nernst and Hill plots for hMb show no cooperativity, as expected for this monomeric system. Nernst plots for Hb A, like the comparable Hill plots, typically have intermediate slopes (n) greater than 1, indicative of cooperative interactions between subunits in the oxidation process. Slopes (n) near unity are observed for Hb at oxidizing and reducing extremes where samples are almost completely oxidized or reduced. The slope of the Nernst plots and the potential at the midpoint of the oxidation curve does not vary with mediator concentration (), but does vary with buffer and background electrolyte concentrations as described below (I).


Figure 2: A, plots of log [oxygenated/deoxygenated] for Hb A and hMb under varied conditions as a function of log oxygen pressure. Experiments at neutral pH, 20 °C. See Table IV for derived parameters and conditions. B, plots of log [oxidized]/[reduced] for Hb A and hMb under varied conditions as a function of applied potential divided by 58.1 according to the Nernst equation. Data obtained from spectroelectrochemical measurements at neutral pH, 20 °C. See Table III for derived parameters and conditions. Symbols: open circles, hMb, 0.05 M MOPS, 0.2 M NaNO; closed circles, Hb A in 0.05 M MOPS; open triangles, Hb A in 0.05 M MOPS, 0.2 M NaNO; closed triangles, Hb A in 0.05 M MOPS, 0.2 M NaNO, 16-fold excess IHP.



The functional properties of Hb A are very sensitive to buffer constituents, particularly those that have the potential for interacting with anion-binding sites on the Hb tetramer. For Hb A, the potential at half-oxidation (E) and the oxygen pressure necessary for half-saturation (P) were found to be sensitive to buffer and background electrolyte concentration. As shown in Tables III and IV, the oxygen affinity of Hb A is significantly lower in 0.2 M MOPS than in 0.05 M MOPS. This buffer effect is, however, small compared to the effects of 0.2 M Cl or NO. The anions Cl and NO are shown to be allosteric effectors which stabilize the T state. Fig. 2and Tables III and IV illustrate a shift to a more positive redox potential and higher P values on addition of either of these two heterotropic ligands to Hb. The nitrate anion is a somewhat stronger modulator than chloride at 0.2 M in both oxygen binding and anaerobic oxidation processes. Cooperativity in the oxidation process, as in the oxygenation process, is clearly decreased when the T state is stabilized by addition of NO or Cl. I includes the parameter En which, as defined in Fig. 1, is a quantitative measure of the asymmetry of the Nernst plots. As can be seen from I, addition of heterotropic ligands that stabilize the T state also increase En.

Hb A in the presence of IHP is more strongly shifted toward the low-oxygen affinity T state than with either 0.2 M Cl or NO. This is illustrated in Fig. 2 and Tables III and IV where the potential required for half-oxidation of HbA is shifted to a more positive value and the oxygen tension necessary for half-saturation increases.

We made use of HbCPA as a model of R state hemoglobin. In previous oxygen-binding studies it was shown that HbCPA models R state Hb function (Antonini and Brunori, 1971). As previously reported, the digested protein is non-cooperative, is more readily oxidized than Hb A, and has higher oxygen affinity (Antonini and Brunori, 1971). As shown in Tables III and IV, its redox potential and oxygen affinity are similar to those of R state Hb A.

At a concentration of 0.2 M, nitrate has little or no effect on the oxygen affinity of hMb, where no quaternary effects are possible. In contrast, nitrate shifts the quaternary conformation of Hb A toward the T state, with concomitant shifts in redox potential and oxygen affinity. Nitrate effects on hMb, HbCPA, Trout I Hb, and Hb A were compared, with the results tabulated in Tables III and IV. As shown in I, the nitrate dependence of Hb redox chemistry is in the order: HbCPA (little affected) < Trout I Hb < Hb A (most affected). This order of sensitivity is consistent with shifts in oxygenation parameters () and the known structural differences among these proteins.

Trout I Hb, Hb A, and HbBv in the absence of heterotropic ligands exist in an R/T equilibrium intermediate to the extremes of HbCPA (R state stabilized) and HbA/IHP (T state stabilized). Their order of increased equilibrium shift toward the R state is HbBv < Hb A < Trout I Hb (Antonini and Brunori, 1971), which is consistent with the observed shift to more negative E values (increasing ease of oxidation; see I) under identical conditions.

Cooperativity

The initial stages of both oxidation and oxygenation curves depict the behavior of Hb when the deoxy Fe(II) condition is dominant. In the T state conformation, the tetrameric state is highly favored (Antonini and Brunori, 1971; Imai, 1982) so that dissociation effects do not complicate this phase of the curves. As previously reported, increases in strength of anionic effectors generate a family of apparent ``T states'' in the oxygenation curves, characterized by decreased oxygen affinity even at very low (<10%) levels of oxygenation (Imai, 1982). provides data on the effects of anions and buffers on the oxygen affinity at 10% oxygenation as well as at 50% oxygenation to document this effect under the conditions used in this study. As a result of this effect, it takes considerably higher levels of oxygen to bring Hb A to 10% saturation with oxygen in the presence of IHP (or IHP with nitrate) than in the presence nitrate alone. In contrast, at low levels of oxidation (<10%) the redox potential is not shifted by IHP over that observed in 0.2 M nitrate or chloride. As will be discussed, we consider that the difference in the initial stages of the oxidation and oxygenation curves is attributable to IHP-induced steric changes in the active site region that hinder oxygenation, while not altering the ease of electron transfer.

Cooperativity between subunits results in slopes (n) greater than one in Nernst plots. Fig. 3summarizes the observed relationship between maximum cooperativity (n) and redox potential for the test proteins studied under varied experimental conditions. The results obtained are in general agreement with predictions of the two-state model (Rubin and Changeux, 1966), with maximal cooperativity exhibited in oxidation of those systems of intermediate redox potential, where neither R nor T states are strongly stabilized. Proteins and conditions where the E values are more positive than observed for the maximum n value in Fig. 3 (E > -100 mV) represent cases where the T state is stabilized relative to R and the degree of cooperativity, represented by n, decreases. Those proteins and conditions where the E value is shifted negatively from the maximum n value in Fig. 3 (E < -100 mV) represent systems where the R state is stabilized relative to the T state and the degree of cooperativity, represented by n, decreases. The shape of the curve shown in Fig. 3 is not adequately described by the bell-shaped curve predicted by the simple two-state model (Monod et al., 1965). We speculate that this is due to the influence of chain heterogeneity on the oxidation curves (see below).


Figure 3: Plot of n as a function of E for various hemoglobins in the presence and absence of heterotropic ligands at pH 7, 20 °C. Data are from Table III. Data points: 1, Hb A in 0.05 M MOPS, 0.2 M NaNO, and 0.25-0.6 mM IHP (in excess over [heme] which varied from 0.1 to 0.23 mM); 2, Hb A in 0.05 M MOPS, 0.2 M NaNO; 3, Trout I Hb in 0.05 M MOPS, 0.2 M NaNO; 4, Hb A in 0.2 M MOPS; 5, Hb A in 0.05 M MOPS; 6, HbCPA in 0.05 M MOPS, 0.2 M NaNO, and 0.25-0.6 mM IHP (in excess over [heme]); 7, HbCPA in 0.05 M MOPS, 0.2 M NaNO; 8, HbCPA in 0.2 M MOPS; 9, hMb in 0.05 M MOPS, 0.2 M NaNO.



When either the R or T state is strongly stabilized, it becomes possible to observe chain differences that are reflected by slopes of less than 1 in Nernst plots. These are observed at low levels of oxidation for HbCPA (R state stabilized) and Hb A + IHP (T state stabilized). Since oxygen-binding curves for these proteins do not show this effect, we conclude that under these conditions the redox potentials for the chains are more distinctly different than are their oxygen affinities. This conclusion has been reached by other researchers, using different methods (Edelstein and Gibson, 1975). A separate study of chain differences using our spectroelectrochemical methods to examine the behavior of mixed metal hybrids will be published elsewhere.()


DISCUSSION

Results reported here allow us to compare oxidation curves for hemoglobin to oxygenation curves obtained under similar experimental conditions. The spectroelectrochemical method utilized allows us to quantify anion effects on the oxidation curves more readily than was previously possible. To a first approximation, the observed E value is a specific measure of the overall Fe(III)/(II) equilibrium, while n gives an indication of the conformational shift between T and R states during the process of oxidation. Differences between n and n reflect the asymmetry of the system under investigation. We find that the asymmetry that gives rise to the difference between n and n is very sensitive to factors that influence the shift between high and low-oxygen affinity conformations. The greater ease of oxidation of HbCPA relative to Hb A, confirmed herein by our results, was an important prior result (Antonini and Brunori, 1971) with regard to establishing that the R state and T state conformations of Hb A have different redox potentials. For HbCPA we find n values of 1 in the mid-range of the oxidation curve and n < 1 in the initial stages of oxidation. Values of n < 1 are taken to be an indication of chain heterogeneity.

In general, as conformational shifts occur, there are changes in both redox potential and oxygenation affinity. As previously reported, the general trend is that the low-oxygen affinity T state is much less susceptible to oxidation than the high oxygen-affinity R state. As was also found by previous investigators, anionic effectors that stabilize the T state typically make the protein less susceptible to oxidation (Antonini and Brunori, 1971). Although our results support these general trends, our methodology allows us to probe interesting situations where shifts in oxygen affinity are not correlated with shifts in values of the redox potential.

One of the striking findings from this study is that anions can affect oxidation and oxygenation differently. As shown in Fig. 2, oxygenation curves are much more sensitive to anions in the initial stage of the process than are the oxidation curves. It is evident from these data that there are anion-linked effects that impede oxygen binding to the T state that are not reflected in oxidation of the T state.

Our results converted relative to the normal hydrogen electrode (NHE) for Hb A are E = +137 mV, n = 1.5, at 20 °C in 0.05 M MOPS, pH 7.1, with 0.2 M NO as a background electrolyte. At 30 °C, Antonini and co-workers(1964) reported E = +150 mV (NHE) and n = 1.5 for Hb A in phosphate buffer at pH 7.0. Similar values were obtained in the pioneering work of Taylor and Hastings(1939) who reported E = +150 mV (NHE) and n = 1.5 for Hb A in 0.2 M acetate buffer at pH 6.8 with a ferrocyanide mediator. Brunori and co-workers(1964), using similar methods and conditions, reported E = +60 mV (NHE) and n = 1 for HbCPA. Under our experimental conditions we find E = +45 mV (NHE) and n = 1 for HbCPA. We find in our laboratory that working at the conditions of Brunori and co-workers(1964) Taylor and Hastings(1939), and Antonini and co-workers(1964) that potentiometric titrations in stirred protein solutions with a ferricyanide mediator result in a shift of the redox potential to a more positive value by about 15 mV. This may be due to increased autooxidation of the protein in stirred solution and/or the allosteric effect of ferri/ferrocyanide.

Comparative oxygenation and oxidation parameters reported in Tables III and IV for Hb A are consistent with previous reports found in the literature. Where differences exist, they can be readily ascribed to differences in ionic strength, mediators, background electrolyte, pH, and temperature. The most critical differences in experimental conditions are the mediator and the background electrolyte, either or both of which can act as allosteric effectors or denaturants. Notably, the redox active dyes most commonly used in previous methodologies are themselves anionic effectors. Additionally, in most previous studies the supporting buffer was typically 0.1-0.2 M phosphate buffer. Since phosphate is a strong anionic effector, its use precludes observation of many anion-linked effects.

Most of the previous studies of Hb A oxidation have not used spectral analysis in combination with measurements of redox potential. Our spectroelectrochemical methods offer this advantage. The use of an OTTLE cell provides a number of other advantages, including improved control of the solution potential and a very small working solution volume (<500 µl), both of which result in rapid bulk electrolysis. The small working volume also precludes the necessity for solution stirring, which therefore lessens the tendency for protein denaturation and autooxidation.

Previous spectroelectrochemical studies of the redox behavior of Hb A that made use of an anaerobic OTTLE cell produced variable results that depended on Hb concentration, the identity of the mediator, and the wavelength selected to monitor the redox process. When Hb A was examined at pH 7.0 in phosphate buffer with 0.2 M KNO using methylene blue as a mediator, a formal redox potential of -160 mV (Ag/AgCl) and n = 4 was reported (Song and Dong, 1988). When a cresyl blue mediator was used as a mediator a formal redox potential of -81 mV (Ag/AgC1) was found using 550 nm to monitor the reaction and -67 mV (Ag/AgCl) when 405 nm was used; the n value reported was 1.4 (Dong et al., 1989). These observations strongly suggest interactions between Hb A and the mediators used. By using Ru(NH) as a mediator, we eliminate these complications.

Our understanding of the molecular controls of the oxygen affinity and oxidation of hemoglobin remains incomplete in spite of many advances in the field that have shown both proximal and distal side effects on the reactivity of the heme iron. There are clearly many features that contribute to the function of the active site. Strong effects on binding of ligands to the heme can be exerted by steric hindrance of axial ligation, local polarity, and hydrogen bonding to the bound ligand. Studies of genetically engineered hemoglobins and myoglobins and low molecular weight analogues have demonstrated that combinations of these factors can significantly alter the relative affinity of heme iron for specific ligands, with varied effects on redox potential (Robertson et al., 1994; Chang and Traylor, 1975; Geibel et al., 1978; Traylor and Traylor, 1982; Suslick et al., 1984; Brantley et al., 1993; Carver et al., 1992; Nagai et al., 1987; Mathews et al., 1989). The use of site-directed mutagenesis techniques in relation to Hb function has focused on modifications of the distal histidine (E7) and valine (E11) residues that alter steric hindrance and/or polarity in the heme pocket (Nagai et al., 1987; Mathews et al., 1989).

Comparison of redox properties of hemoglobin with oxygenation properties can help differentiate between steric and electronic control mechanisms. The redox potential is clearly responsive to the electron-density alterations that result from T to R state conformational shifts. In the studies reported herein, deoxyhemoglobin, in which the Fe(II) is five-coordinate, is oxidized to metHb, in which the Fe(III) adds a sixth ligand, HO. This HO ligand is readily available in the heme pocket, and the available evidence suggests that HO has no steric influence on the redox process (Perutz et al., 1987).

In addition to electronic control of the active site, functional effects specifically attributable to steric hindrance have been clearly demonstrated in both model compounds and heme proteins by comparison of the binding of CO with more bulky isocyanides (Olson, 1976; Reisberg and Olson, 1980; Traylor et al., 1980). From both theoretical and experimental considerations, a change in the stereochemistry of the active site, brought about by an allosteric effector, could alter active site ligand affinity without altering the oxidation potential. Studies with active site analogues have shown that steric influences can result in a functional discrimination between ligands, such as oxygen and carbon monoxide, without having an appreciable effect on oxidation potential (Traylor and Traylor, 1982; Perutz, 1990).

To consider the results in more detail and come to a better understanding of the physical basis of the differential effect of IHP that we observe in the initial stages of the oxygenation and oxidation curves, we must take into account the differences between oxygen affinities and the oxidation potentials of the and chains. Perutz (Antonini and Brunori, 1971) proposed on the basis of x-ray structural data that steric effects would result in preferential binding of oxygen with the chains of T state Hb. This was subsequently verified by functional studies that showed chain heterogeneity in both oxygenation and oxidation. The preferential oxidation and oxygenation of chains is enhanced by the presence of strong allosteric effectors such as IHP (Brunori et al., 1968; Perrella and Rossi-Bernardi, 1981; Perrella et al., 1990; Di Cera et al., 1987; Banerjee and Cassoly, 1969). Using NMR techniques, Johnson and Ho(1974) found almost exclusive oxygen binding to the chain hemes in the presence of IHP. Previous researchers have drawn the conclusion that in the deoxygenated condition the chains are preferentially oxygenated and preferentially oxidized. Using a rapid-mixing methodology, Edelstein and Gibson(1975) estimated that the chains have a 2-fold lower affinity for electrons than the chains in the absence of phosphates, and at least a 4-fold lower affinity in the presence of IHP. Thus, the initial stages of oxidation and oxygenation as shown in Fig. 2and Fig. 3may be considered to be dominated by the behavior of the chains.

Although the results cited above strongly suggest that chains are preferentially oxidized and oxygenated in the initial stages of these processes, the Nernst plots show that chain heterogeneity is still appreciable. The initial stages of oxidation in many cases show n values significantly less than one, indicative of more than one type of site being oxidized, or coupling between sites that results in negative cooperativity. In the presence of high levels of IHP, n values less than one are manifest throughout the oxidation process, showing that the process cannot at any time be associated with a single type of chain unless one invokes the possibility of negative cooperativity between sites.

We speculate that the differential effect of IHP that we observe in the initial stages of the oxygenation and oxidation curves is due to steric factors that impede oxygen binding. We rule out changes in proximal strain as the underlying cause since these would clearly affect the redox state, as shown by redox changes that accompany the R to T transition. Similarly, if changes in the polarity in the active site accompany IHP addition, one would expect an alteration in redox state as well as in ligand affinity. Two possibilities remain. As IHP is bound, there could be a decrease in hydrogen bonding to the bound oxygen that would result in decreased oxygen affinity, thereby altering the initial stages of oxygenation without affecting the comparable part of the oxidation curves. This mechanism would require that hydrogen bonding to liganded oxygen occurs to an appreciable extent and that this is disrupted by IHP. The second possibility, which we favor, is that a steric hindrance effect arises as a result of IHP binding. This would require allosteric stabilization of a sterically hindered state or movement of distal residues toward the heme iron. Heme pocket alterations in response to IHP addition are supported by NMR studies on oxy and CO forms of Hb (Lindstrom and Ho, 1973), but comparable data on the deoxy form are not available.

In conclusion, results obtained by a spectroelectrochemical method using Ru(NH) as a non-allosteric mediator allow us to compare anion effects on the oxidation of hemoglobin with anion effects on the oxygenation and autooxidation processes. Anions shift the R/T equilibrium toward the T state. This causes common trends in the anion-linked shifts in oxygen affinity and oxidation potential that can be accounted for in terms of the two-state model and the equilibria of . However, there are striking differences between IHP effects on the Nernst and Hill plots, particularly in the initial stages of the process (see, for example Fig. 2). These differences make it clear that anions can, in some cases, affect ligand binding without significant alteration of the redox potential.

Differences between redox and oxygenation curves show that hemoglobins have, by nature's design, used stereochemical mechanisms to offset or mediate the redox potential of the active site. This insight, although not new to the field of Hb structure-function relationships, may encourage the design of synthetic hemoglobins or active site mimics that use similar steric control mechanisms to modulate function. We anticipate that the spectroelectrochemical techniques introduced here will prove valuable in differentiating between steric and electronic effects in the many new Hbs and Mbs that are becoming available for study as a result of protein engineering, as well as in natural Hbs and Mbs from varied sources where environmental and physiological pressures have led to novel mechanistic solutions to the problem of controlled oxygen delivery without excessive oxidation.

  
Table: Spectroelectrochemical results for simple electroactive systems


  
Table: 0p4in Buffer/electrolyte 0.05 M MOPS, 0.20 M KCl, pH 7.1.(119)

  
Table: Spectroelectrochemical data for hMb, Hb A, -SH, HbBv, Hb Trout I, and HbCPA in the presence and absence of heterotropic ligands


  
Table: Oxygen binding for hMb, Hb A, and HbCPA in the presence and absence of heterotropic ligands



FOOTNOTES

*
This work was supported by the Duke University Arts and Sciences Research Council and technical services and facilities were made available by NIEHS Center Grant ESO 1908. The costs of publication of this article were defrayed in part by the payment of page charges. This article must therefore by hereby marked ``advertisement'' in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.

§
To whom correspondence should be addressed.

The abbreviations used are: IHP, inositol hexaphosphate; MOPS, 3-[N-morpholino]propanesulfonic acid; DCPIP, dichlorophenolindophenol; OTTLE, optically transparent thin layer electrochemical cell; NHE, normal hydrogen electrode.

K. M. Faulkner, C. Bonaventura, B. M. Hoffman, and A. L. Crumbliss, manuscript in preparation.


ACKNOWLEDGEMENTS

We gratefully acknowledge helpful discussions with Dr. Fethi Bedioui, Ecole Nationale Supérieure de Chimie de Paris.


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