Journal of M-r Liquid& 42 (1969) 167-174 j3sevier Science Publiahere B-V., Amstmdam -Printed in The NekherIands

167

THE USE OF A SUPERCONDUCTING MAGNBTOMBTBR TO MBASURB SPIN

EQUILIBRIA IN HBMOGLOBIN

Massimo Cerdonio*, Silvia Morantes, St%fano Vitale*, Alice DeYoung+, and Robert W. Noble+

*Dipartimento di Fisica, Universita di Trento, POVO (Trento), Italy SDipartimento di Fisica, II Universith di Roma "TOT Vergata", Roma, Italy +Departments of Medicin% and Biochemistry, SUNY at Buffalo, V-A. Medical Center, Buffalo, N-Y- 14215, U-6-A.

We are very glad to have the opportunity to outline the outcome of so many years of happy collaboration in this volume to honour Prof, Giorgio Careri, Indeed it is quite appropriate to do 80. Even if th% reatler will not find referenced any paper in collaboration with him, still his role in this work has been just as great and rel%vant,

It iti thanks to Prof. Careri that on% of us (M-d,) was at first introduced to the world of superfluidity and superconduativity and then to biophysica-

Superconducting electronics, with SQUIDS %tc,,was offering good opportunities for the developement of ausceptometers sensitive and ac curate enough for biological studi%s, The first such efforts were carried out in J.im Merc%reau's laboratory in Caltech hy E. Hoenig and Run Han Wang. One of the authors, M-C-, following the style of Caretri'a laboratory, had the opportunity t0 spend one year doing research abroad in an outstanding laboratory- As he had been using SQUID8 for high resolution magnetic studi%s in solid state physics and wanted to expand his studies to biological matter, hb chose to go to Caltech, where he also developed his interest 5-y. hemoglobin, Afterwards, in Careri's laboratories in Rome and Monterotondo, SQUID susce

P tome':ers were developed, with which the authors carried out

the f rst uxperiment on ferric hemoglobin with Max Perutz, These studies have been pursued up to the present, %v%n after M,C,‘s laboratory moved to Trento. Therefore, we would lik% to thank Prof, Giorgio Careri for creating the opportunity by which w% have been able to enjoy this collaboration for all these years.

Transition metals play a central role in a variety of

processes in biological systems~ In general they are found

coordinated to a variety of nucleophilic ligands such as the

nitrogen of the imidazole ring of a histidine residue or the

oxygen of a carhonyl or carhoxyl group- In the course of their

functional activities they are frequently found to change their

oxidation stat%, their coordination state or- both- A prime

example of the latter is the iron atom as it exists ?n

0167-7322/88/$63-= 6 1sssElsevierScien~PublishereB.V.

168

hemoglobin. This iron can occur in either ita ferrous or ferric

oxidation stats and is tightly coordinated with the fOW

nitrogens of the pyrrol rings of the porphyrin ring. It is also

coordinated with an imidazole nitrogen of a histidine residue of

the protein. Its sixth coordination site is available for the

binding of exogenous ligands. When the iron is in its ferrous

oxidation state, it can bind oxygen (its physiologically relevant

ligand) aa well as carbon monoxide, nitric oxide and a number of

other compounds, In its ferric state it binds among other

things, cyanide, azide, and nitrite; and in the absence of any

other ligands it binds water. Thus, in the ferric state the

sixth coordination po&tIon is always occupied.

It was pointed out by Kotani that in hemoglobin the iron

atom is poised mkween two electronic configurations (for ~1

review see (1)). The sixth ligand can alter the energy 6plitting

between the d orbitals thereby altering the equilibrium between

the high SPin and low spin electronic statea, Thus, the

ferrous, deoxygenated heme is high spin, butthe binding of

oxygen raises the energies of the d,2-y2 and dz2 orbitals

relative to those of the dxy, dxz and dyz orbital8 to such an

extent that essentially only the latter are populated, In the

f43JZCiC state the presence of a weak ligand such as water or

fluoride results in little or no energy splitting 60 that to a

first approximation the 5d electrons are entirely unpaired,

resulting in the high spin, S=5,f2 state, On the other hand, the

binding of cyanide ion yields the fully low spin, s-1/2 state.

Other ligands result in a variety of energy splitting6 and

equilibria between spin states.

High and low spin stats8 display a number of diagnostic

spectral features, However, the most direct means of

quantitating spin states and equilibria of hemoglobin derivatives

is the determination of their magnetic susceptibilities. Most of

the studies of paramagnetism of iron in hemoglobin were made

using the temperature dependence of susceptibility according to

the curie law. This requires measurements over a wide

temperature range so that most of the measurements had to be

carried out in the frozen state. This poses a number of

problems, Normally the hemoglobin ruolecule functions in aqueous

solution, ItfI propertiee are strongly pH dependent and defining

this parameter in the frozen state is difficult, Derivatives

which exhibit intermediate spins have temperature dependent spin

equilibria resulting in an inverse curie dependence. At very low

temperatures the activation energy for the spin state transition

b@LCOIllEU3 much greater than WC, essentially freezing in a spin

distribution. As demonstrated by Iizuka and

in magnetic properties which depend upon the

temperature is lowered.

For this reason the kind of variable

Kotani this retiulta

rate at which the

temperature, high

resolution, cryogenia SQUID susceptometers developed at first in

Rome and Monterotondo and afterwards in Trento turned out to be

the best instruments for these studies, The earliest versions

were still somewhat cumbersome in operation and calibration (3-

51- The latest version which started operation in Trento in 1978

and is still working today is truly a routine operation

instrument (6). The main features are the following. The full

room temperature range can be explored, from just above the

freezing point of the solution up to temperatures where protein

denaturation begins, These conditions can be maintained for sets

of measurements taking as long as one needs, since the helium can

be refilled without disturbing the sample and without altering

the calibration or any other conditions of the measurement. The

amount of sample needed for each measurement is about 0.5 ml and

the sample can be completely recovered. Typical concentrations

in heme for hemoglobin studies ranged from 2mM up to 15mM. A

single set of measurements on one sample requires less than one

hour on order to obtain the highest resolution of 0,0003 of the

susceptibility of water. This permits comparisons with blanks

and between samples with different solution conditions all in a matter of a few hours. The limiting factor for the accurancy in

getting the iron magnetic moment of a 3mM in hema hemoglobin

solution is rather the error in the determination of the

concentration itself-

This apparatua has been applied to the study of two aspects

of the biophysics of the hemoglobin molecule, One has been an

examination of the extent to which the liganded forms of ferrous

hemoglobin are fully low spin, ie, fully diamagnetic. The other

has been as examination of the influence of the conformational

state of the protein on the spin equilibria of mixed spin, ferric

derivatives,

Ferrous, deoxygenated hemoglobin binds oxygen, as well as

its other ligands, cooperatively, This increase in ligand

170

affinity with increasing saturation arises as a result of a

lAgand linked change in the quaternary structure of the protein

from a low affinity,T state generally associated with the

deoxygenatad molecule, to a high affinity, R state which is

observed with the ligand saturated molecule, A simple model for

cooperativity invoking only these two conformational states was

presented by Monod, Wyman and Changeux (71, Although

intermediate structural states may wall occur at partial

saturation With ligand, this "two state model" represents a

reasonable first approximation to the properties of the

hemoglobin molecule, For OUT proses it i6 Of 6peOial Utility

since it serves to focus attention on a central question which we

have been exploring, ie. what is the physical or structural basis

for the difference fn the ligand affinities of these f3tructura1

states?

The orfgin of this affinfty difference has been the topic of

much speculation and experiment. One possibility which was

suggested some time ago is steric interference on the distal side

of the heme, where the ligand binds, resisting the proper

positioning of the ligand at the heme iron (8). This possibility

was examined extsneively by Him8 et al-(g) and by Lin et al.(lO).

They studied the binding of a variety of isonitriles, which vary

fn bulk or size, and they did not find the relationship between

ligand size and the affinity difference which would be expected

if this mechanism contributed significantly to the difference In

the ligand affinities of the R and T states.

A very different mechanism for the R and T state difference

in ligand afffnity was postulated by Perutz (11). In

deoxygenated hemoglobin the Iron atom lies outside the plane of

the porphyrin, being shifted in the direction of the proximal

histidine residue by about half an Angstrom (12). The binding of

oxygen or carbon monoxide is accompanied by a shift of the iron

to a position within the porphyrin plane- This movement of the

iron atom results in a movement of the proximal histidine

imidazole ring toward the heme. Perutz suggested that it is this

movement which is resisted more in the T state than in the R

state.

This model, as first formulated, led to the expectation that

the resistence to motion in the T state would result in a strain

or tension directly at the here which would evidence itself by

171

changes in bond energies, and thereby by changes in the resonance

frequencies of the bonds, Comparisons of the R and T states of

liganded derivatives of hemoglobin by IR and resonance Raman

spectroscopy reveal no- such differences (X3), -In the

deoxygenated derivative a difference was observed between the

stretching frequencies of the bond between the iron and the

proximal histidine in the R and T states (14,15). However, the

energy associated with this frequency change is small compared to

the energy of the affinity change which we seek to understand,

although the frequency change is in the correct direation (16).

This situation is consistent with the suggestion of Hopfield (17)

that most UP the energy of cooperativity is distributed

throughout the protein but could still be coupled to the motion

of the iron atom, This would explain the failure to observe it,

but can hardly be used to establish the correctness of the model,

How can one demonstrate whether or not the conformational

transition of the protein from the T to the R state is

thermodynamically coupled, or sensitive, to the positioning of

the iron and proximal histidine residue with respect to the plane

of the hetme? Examining the effects of this conformational

transition on the eguilibrium between tha deoxygenated ancP

liganded ferrous derivatives of the molecule, ie- ligand

affinity, could not give an answer since this reaction involves

not only a change inthegeometry of the heme-histidine system

but the attachment of a ligand at the previously unoccupied c&th

coordinate position of the iron- Instead one needs to examine

the effects of protein conformation on a process which as nearly

as possible involves only a change in geometry without a change

in lignnd binding. It was Max Perutz who first realized how to

approach this problem- As already mentioned, many ferric

derivatives of hemoglobin exist as mixtures of high and low spin

states which are in equilibrium with one another. From the work

of Hoard (18) on model aompounds it is well accepted that in

general low spin coordination complexes of iron are characterized

by shorter bond distances than are high spin aomplexes.

Therefore, it is expected that the high spin states of ferric

hemoglobin derivatives will have longer bonds between the iron

and the five fixed nitrogen liganda than the low spin states,

This confers on them a le6s rigid structure, permitting a greater

movement of the iron with respect to the heme plane, This being

so, if the Perutz model were corraat, then the spin equilibria of

172

ferric derivatives of hemoglobin should be sensitive to protein

StZUCt~e with the high spin state being energetically more

favorable in the T state than in the R state.

One additional detail had to be dealt with before the

experimants could be successfully undertaken, It was obviously

necessary to be able to reversible switch derivatives of ferric

hemoglobin from the R to the T Etate- penltz et a11(19) had

reported evidence that the compound inositol hex&phosphate, IHP,

could convert predominantly high spin derivatives of human ferric

hemoglobin to their T states, but could not do the same for lower

spin derivatives such as the azide form_ However, one of us

(RWN) had been studying the hemoglobin of the carp for many years

and had shown that this hemoglobin, as well as othar fish

hemoglobins, could be switched to its T state at pH 6 in the

presence of organic phosphates even when fully low spin (2O,21),

Carp hemoglobin became an important model system for our studies,

Beaause of the low concentration of iron atoms in

hemoglobin, the diamagnetism of the protein and the aqueous

solution in which it is dissolved is far greater than the

paramagnetia effects of even the high spin iron atoms.

Therefore, in order to determine the paramagnetic susceptibility

of a sample, its diamagnetic properties must be precisely

defined, To this end we determined the gram susceptibility of

protein to be -0.587 x lO+ml/g (22)- This, along with precise

measurement of the diamagnetic susceptibility of each buffer

system used in our studies, permitted the determination of the

paramagnetic properties of our samples.

Our results with carp hemoglobin can be stated quite sfmply.

The conformation of this hemoglobin molecule has profound effects

on the spin equilibria of all of the ferric derivatives which we

examined (23,24)- The most dramatic change in susceptibility in

response to the R toT state conformational transition is

observed with the azide derivative. This is due to the fact that

the concentrations of the two spin states of this derivative are

most nearly equnl at equilibrium_ The change in the standard

frae energies of the spin equilibria which accompany the change

in protein conformation average roughly 700 Cal/mole, between 20

and 25% of the free energy of cooperativity of the binding of

ligands to ferrous, deoxygenated hemoglobin-

One may wonder why identifying only a fraction of the energy

173

of cooperativity can be considered to be a successful experiment.

However, this iti precisely the result which one woulip expect from

the Penltz model. Although high spin ferric derivatives permit

more iron motion than low spin cerivativss, they offer far less

flexibility than that found in the pentacoordinate, deoxygenated

ferrous state- The absence of a sixth ligand allows much greater

geometrfcal distortion than can be achieved when one in present,

even when the bond tc it is relatively long. No displacements of

the iron atom from the heme plane have been found in liganded

derivatives that approach that observed in deoxygenated

hemoglobin-

As satisfying as the data for carp hemoglobin were, it was

Still unclear if the effect observed was a general one, or

peculiar to carp or perhaps to fish hemoglobins. Thk issue was

focused upon directly by the report of Phi10 and Dreyer (25) that

the effect of IHP on the spin equilibria of predominantly high.

spin forms of human ferric hemoglobin is much smaller than the

effects we observed with carp hemoglobin. We postulated that the

data of Philo and Dreyer were correct, but not their

interpretation. Specifically, we questioned that IRP was truly

causing a complete transition to the T state of any liganded

derivative of human hemoglobin, We tested this hypothesis by

examining the combined effects of IHP and a second compound,

bezafibrate, This compound binds preferentially to the T state

of hemoglobin but at a different site than IRP, so that the

effects of the two effector molecules are synergistic (26,27).

Their combined effects on the spin equilibria of derivatives of

ferric human hemoglobin proved to be in good agreement with the

effect of the R to T state transition in carp hemoglobin (28) -

Therefore, we have concluded that this is a general effect in

hemoglobin, not a paculsrity of one particular hemoglobin

molecule-

As we have noted previously, these data offer no direct

insight into the actual mechanism by which the relative energies

of the high and low spin electronia statas are affected by the

structural state of the protein. However, they clearly establish

a linkage between the state of the iron atom and the 8trUCtUre of

the protein which is consistent with the model we wish to test.

Furthermore, this property of liganded Derivatives of hemoglobin

reinforces the resonance Raman studies of deoxy and photolysed

174

ferrous derivatives which implicate the proximal histidine and

its bond with the iron atom in the th@J3IlOdpXlIUiC linkage of

structural changes in the protein to the chemistry of the heme.

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