From the
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.
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
On-line formulae not verified for accuracy
On-line formulae not verified for accuracy
On-line formulae not verified for accuracy
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
Samples of chromatographically
purified human hemoglobin A
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.
Spectroelectrochemical experiments were
carried out in an anaerobic optically transparent thin layer
electrochemical cell (OTTLE) made of a 1
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
Spectroelectrochemistry was carried out at a single wavelength,
typically one of the protein Soret bands (430 nm,
On-line formulae not verified for accuracy totally oxidized hemoglobin was set to zero (A
On-line formulae not verified for accuracy 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
On-line formulae not verified for accuracy 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
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
Hexammineruthenium(III/II)
(Ru(NH
MOPS buffers, with varied levels of nitrate as the
background electrolyte, were found to work well in electrochemical
experiments with Ru(NH
The
E
Fig. 2
compares Nernst and Hill plots for the oxidation and
oxygenation of hMb and Hb A
Hb A
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
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
Trout I Hb, Hb A
Cooperativity between subunits results in slopes
(n) greater than one in Nernst plots. Fig. 3summarizes
the observed relationship between maximum cooperativity
(n
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
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
Comparative oxygenation and
oxidation parameters reported in Tables III and IV for Hb A
Most of the previous
studies of Hb A
Previous spectroelectrochemical
studies of the redox behavior of Hb A
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, H
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
Although the results
cited above strongly suggest that
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
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.
We gratefully acknowledge helpful discussions with Dr.
Fethi Bedioui, Ecole Nationale Supérieure de Chimie de Paris.
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
FOOTNOTES
ACKNOWLEDGEMENTS
REFERENCES
T equilibrium shift associated with both the oxygenation and redox
processes.
(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.
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.
(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.
Oxygen Binding and
Spectroelectrochemistry
Oxygen equilibria measurements
were performed tonometrically, with the spectrophotometric method of
Riggs and Walbach(1956).
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.
)
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.
= 1.33
10
M
cm
for deoxyHb and 406 nm,
= 1.62
10
M
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
= 0), Equation 1 simplifies to Equation 2.
is
`, 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.
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.
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.
)
), 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.
)
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.
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).
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.
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.
, 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.
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.
, 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.
) 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.
(
)
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.
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.
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.
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.
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.
O. This H
O ligand is readily
available in the heme pocket, and the available evidence suggests that
H
O has no steric influence on the redox process (Perutz
et al., 1987).
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.
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.
)
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.
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
©1995 by The American Society for Biochemistry and Molecular Biology, Inc.