Rotational Correlation Times of3-Carbamoyl-2,2,5,5-tetramethyl-3-pyrrolin-1-yloxy Spin Label

with Respect to Heme and Nonheme Proteins

S. Cavalu*,† and G. Damian‡

Faculty of Medicine and Pharmacy, Department of Biophysics, University of Oradea,P-ta 1 Decembrie No. 10, Oradea, 3700, Romania, and Faculty of Physics, Babes-Bolyai University,

Ro-3400 Cluj-Napoca, Romania

Received March 26, 2003; Revised Manuscript Received August 26, 2003

Noncovalent spin labeled proteins (ovalbumin, bovine serum albumin, hemoglobin, and cytochromec) wereinvestigated in order to follow the different type of interactions between the nitroxide radical of 3-carbamoyl-2,2,5,5-tetramethyl-3-pyrrolin-1-yloxy spin label and functional groups of heme and nonheme proteins aswell as the pH influence on molecular motion of the label with respect to these proteins. EPR spectra wererecorded at room temperature and the computer simulation analysis of spectra was made in order to obtainthe magnetic parameters. Noncovalent labeling of proteins can give valuable information on the magneticinteraction between the label molecule and the paramagnetic center of the proteins. The relevance of thisinteraction can be obtained from line shape analysis: computer simulations for nonheme proteins assumea Gaussian line shape, whereas for heme proteins, a weighted sum of Lorentzian and Gaussian componentsis assumed. In the framework of the “moderate jump diffusion” model for rotational diffusion, the rotationalcorrelation time is strongly influenced by pH, because of the electrostatic interactions and hydrogen bonding.

Introduction

The successful application of spin labeling to proteinstructure investigations is limited by the possibility tochemically change specific side chains in proteins. However,useful information on protein properties can be obtained bynoncovalent spin labeling if the affinity of the protein forthe label molecules is great enough to affect their motionalfreedom.1-5 The rate of rotation (or tumbling) of the spinlabel influences the line shape of its EPR spectrum.Therefore, the EPR signal of a spin label covalently ornoncovalently bonded to a biomolecule can yield a range ofinformation about its structural environment in conventionalESR and allows for full spectral coverage.6,7

At the same time, EPR has been an invaluable tool forprobing microscopic molecular motions in a variety ofsystems, including isotropic solvents,8 liquid crystals,9 modelmembranes, and biomolecules.10-12

In the present work, noncovalent spin labeled bovine serumalbumin (BSA), ovalbumin, bovine hemoglobin (BH), andcytochromec, with Tempyo spin label (3-carbamoyl-2,2,5,5-tetramethyl-3-pyrrolin-1-yloxy) were investigated both inliquid and lyophilized samples, in the pH range 2.5-11, toobtain useful information related to the interaction betweenthe nitroxide group and the functional site of the proteins.

Interactions of the spin label with heme or nonhemeproteins might affect the spin label spectra, and at the sametime, it is well-known that the pH strongly influences the

conformation of proteins leading to significant changes inthe type and degree of these interactions. In this pH range,we followed the effect of protein conformational changeson the interactions between the nitroxide and the functionalgroups of proteins and also the pH influence on molecularmotion emphasized by the EPR spectra of the spin label.

All structural and functional properties of proteins derivefrom the chemical properties of the polypeptide side-chain.The ionizable groups of the side-chains of charged aminoacids are often involved in biochemical transactions (binding,catalysis). Therefore, pH usually has rather dramatic effectson the function of proteins. Polar residues are both buriedas well as on the surface of the protein. They either formhydrogen bonds with other polar residues in the protein orwith water, but nonpolar residues do not interact favorablywith water.

Materials and Methods

Powdered bovine serum albumin, ovalbumin, hemoglobin(>95% methemoglobin) and cytochromec from SigmaChemicals, were used without further purification. Proteinswere hydrated in phosphate buffer physiological saline at afinal concentration of 10-3 M. Tempyo spin-label (3-carbamoyl-2,2,5,5-tetramethyl-3-pyrrolin-1-yloxy), fromSIGMA Chemicals (Figure 1), was added to the liquidsamples of each protein in a final concentration of 10-3 M(protein/spin label molar ratio 1:1) and the pH values wereadjusted to the desired value in the range 2.5-11. A total of5 mL was taken from each sample and was lyophilized for30 h at-5 °C and used for the EPR measurement, at roomtemperature.

* To whom correspondence should be addressed. E-mail: scavalu@rdslink.ro.

† University of Oradea.‡ Babes-Bolyai University.

1630 Biomacromolecules 2003,4, 1630-1635

10.1021/bm034093z CCC: $25.00 © 2003 American Chemical SocietyPublished on Web 10/16/2003

EPR spectra for both liquid and lyophilized samples wererecorded at room temperature with a JEOL-JES-3B spec-trometer, operating in X-band (9.5 GHz), equipped with acomputer acquisition system. Samples were placed in quartzcapillary tubes. The spectrometer settings were as follows:modulation frequency 100 kHz, field modulation 1 G,microwave power 20mW. The computer simulation analysisof spectra, for obtaining the magnetic characteristic param-eters, was made by using a program that is available to thepublic through the Internet (http://epr.niehs.nih.gov)

The line shape of an EPR spectrum depends on, amongother factors, the orientation of the paramagnetic center withrespect to the applied magnetic field. In a powder, or a frozenaqueous solution, the paramagnetic centers will be fixed witha random distribution of orientations, and in the case of theanisotropicg factor and hyperfine interactions, this will leadto a broadened EPR spectrum, because all orientationscontribute equally. In the liquid state, however, the para-magnetic centers are not fixed but undergo rotationalfluctuation. In the case of fast rotation, the anisotropicinteractions are thereby averaged to zero, giving rise to sharpEPR lines. If the velocity of the rotational motion decreases,the EPR spectrum will approach that of the powder spectrum.Therefore, a rotational correlation time for a paramagneticmolecule can also be determined by EPR.

For isotropic motion in the rapid tumbling limit, the spectrawill be isotropic with the averages of the principal compo-nents of theg values and hyperfine splitting factor,aN. Therate of the isotropic motion determines the relative widthsof resonances and the width,∆Hm, of an individual (hyper-fine) line, in the first approximation can be written as afunction of thez component of the nitrogen nuclear spinnumber (m ) -1, 0, 1):13

where theA coefficient includes other contributions thanmotion. The termsB and C are functions related to therotational correlational time (τ) and can be defined as afunction of peak to peak line width of the central line,∆H0

[G], and the amplitudes of themth line Im:11,12

in which

and ωN ) 8.8 × 10-6⟨aN⟩, aN is the isotropic hyperfinesplitting andωe is the ESR spectrometer frequency in angularunits.

In the range from 5× 10-11 to 10-9 s (motion in the rapidtumbling limit) and a magnetic field above 3300 G,∆g and∆aN vanish, and the correlation timesτB andτC are directlyrelated to theB andC coefficients by the following simplerelations:4

whereK1 ) 1.27× 10-9 andK2 ) 1.19× 10-9. The averagecorrelation times is

The slow motion of the spin probe lead to a broadening ofthe EPR lines. In this case, the rotational correlation time,τ, is larger than 10-9 s, and thus, eq 8 is not applicable.

The isotropic nitrogen hyperfine splitting changes to apowder like spectrum, with the peak-to-peak distancebetween the external peaks of the spectrum (2a′zz(N)) de-pending on the magnitude of the rotational correlation time,τ. Another line shape theory for slow isotropic Brownianrotational diffusion of spin-labeled proteins has been devel-oped by Freed and co-workers.8 Thus, the correlation timecan be evaluated from the ratio of the observed splittingbetween the derivate extremaa′zz and principal valueazz,determined from the rigid matrix spectrum:4,6

The R andâ parameters are empirical constants dependingon the kind of the diffusion process and are tabulated in e.g.Poole and Farach.14 For a small spin probe, the intermediatejump diffusion is preferable.14

Results and Discussion

In the low concentration liquid state of proteins solution,the Tempyo spin label, which is a relatively small molecule,gives rise to a spectrum with narrow lines and constanthyperfine splitting, typical for fast isotropic rotational motion(Figure 2) with very low rate of migration between proteinmolecule and water. For this kind of rotation, the rotationalcorrelation time can be estimated from the intensity ratio ofthe low-field and high-field N lines using a semiempiricalformula (eq 8). For the Tempyo spin label in nonhemeproteins (BSA and ovalbumin) aqueous solution, the calcu-lated rotational correlation times was 2.5× 10-10 s, whereas

Figure 1. Chemical structure of the Tempyo spin label.

∆Hm ) A + Bm+ Cm2 (1)

B ) 12∆H0(xI0

I1- x I0

I-1) ) 0.103ωe[∆g∆a

N +

3(δg)(δaN)]τB[1 + 3

4(1 + ωe

2τB2)-1] (2)

C ) 12∆H0(xI0

I1+ x I0

I-1- 2) ) 1.181× 106[(∆a

N)2 +

3(δaN)2]τc[1 - 3

8(1 + ωc

2τc2)-1 - 1

8(1 + ωe

2τc2)-1] (3)

∆aN ) azz

N - 12(axx

N + ayyN ),δa

N ) 12(axx

N - ayyN ) (4)

∆g ) gzz- 12(gxx - gyy), δg ) 1

2(gxx - gyy) (5)

τB ) τz ) K1B (6)

τC ) τx,y ) K2C (7)

τ ) (τBτC)1/2 (8)

τ ) R(1 -a′zz

azz)â

(9)

Rotational Correlation Times of Nitroxide Radical Biomacromolecules, Vol. 4, No. 6, 2003 1631

with respect to heme protein (BH and cytochromec)solutions, the respective value was around 2.× 10-10, whichis consistent with fast rotation as expected for a smallmolecule.

No detectable changes were observed in the EPR spectraof the aqueous solution of the Tempyo spin label withoutproteins, at different pH values.

The characteristic EPR spectra of a Tempyo spin label inlyophilized samples is due primarily to anisotropy in thenitrogen hyperfine coupling typical for slow rotation. Forslow rotations, the EPR spectrum of spin labels, depends ina much more complicated fashion on the combined influ-ences of molecular motion and magnetic interactions. Figures3-5 display the experimental and simulated spectra forTempyo spin label with respect to BSA, ovalbumin, BH,and cytochromec, lyophilized at pH) 2.5, 6.7, and 11,respectively. To find the magnetic parameters, the experi-mental EPR spectra were simulated. The best fit of experi-mental EPR spectra of the Tempyo spin label in lyophilizedBSA and ovalbumin was obtained assuming a singleparamagnetic species and a Gaussian line shape correspond-ing to static dipolar interactions. The magnetic parametersare listed in Tables 1 and 2, respectively.

The main feature of the EPR spectra of the Tempyo spinlabel in lyophilized haemoglobin and cytochromec exhibitsthe characteristics to the slow motion of the spin label butwith more broadening of the line shape. The simulation ofthe experimental EPR spectra can be obtained by assumingthe presence of two functional groups in heme proteins,

associated with two nonequivalent paramagnetic species.15

Computer simulations, indicate a weighted sum of mixedGaussian line shapes (static dipolar interactions) and Lorent-zian line shapes (spin-spin interactions). To support thisaffimation, in Figures 6 and 7 are presented the EPR spectraof the Tempyo spin label in lyophylized BH at low and highpH, in which are displayed the contributions of each species.Generally, the broadening of the Gaussian line shape is dueto the static dipolar interactions of the spin label molecules,whereas the broadening of the Lorentzian line shape is dueto spin-spin interactions.16-17 From the computer analysisof spectra, we suggest that at low pH (pH) 2.5) the maincontributions are due to species with a Gausian line shape(∼80%). This contribution decreases to∼50% at high pH(pH ) 11). The first species, with Gaussian line shape andpoor resolved hyperfine splitting, is not influenced by the

Figure 2. EPR spectrum of the Tempyo spin label at low pH.

Figure 3. EPR spectra, experimental (solid line) and simulated(dotted line), of the Tempyo spin label proteins at pH ) 2.5.

Figure 4. EPR spectra, experimental (solid line) and simulated(dotted line), of the Tempyo spin labeled proteins at pH ) 6.7.

Figure 5. EPR spectra, experimental (solid line) and simulated(dotted line), of the Tempyo spin labeled proteins at pH ) 11.

1632 Biomacromolecules, Vol. 4, No. 6, 2003 Cavalu and Damian

presence of the heme iron, and therefore, we assume to belocated far from the heme group. The second species with aLorentzian line shape and a well resolved hypefine structureis located, probably, near the heme group, giving rise to aspin-spin interaction between the nitroxide radical and theparamagnetic iron of the heme group. Our results are in

accordance with covalently labeled methemoglobin and otherporphyrins in frozen samples under 50 K.18,19 In theseprevious studies, spectra of covalently labeled methemo-globin were analyzed by using perturbation calculations inorder to estimate the iron to nitroxyl distances, and it wassuggested that plausible distances are in the range of 14.5-17.5 Å. Theg tensor andA tensor components used in thesimulation for the best fit values of the simulation of theeffective powder spectrum are presented in Tables 3 and 4(for Tempyo-BH and Tempyo-cytochromec, respectively).

Because of increased spatial restrictions of the proteinstructure in the vicinity of label, by lyophilization, themobility of the spin label is slow on the EPR time scale(∼5 × 106 s-1), leading to a broadening of the EPR lines,with the peak-to-peak distance between the external peaksof the spectrum (2a′zz(N)) depending on the magnitude ofthe rotational correlation time,τ. Generally, a broadeningof the peaks in an EPR spectrum is indicative of immobiliza-tion of the spin label, whereas sharpening of the peaks pointsto an increase in label mobility. By comparison of theapparent nitrogen hyperfine splitting (termeda′zz(N)) withthe nitrogen hyperfine splitting obtained from their rigid limitvalues (a′zz(N)), the rotational correlation times can becalculated using eq 9. The values ofR and â coefficientsdepend on the motional model. The study of the influenceof the different diffusional models on the spectral line shapein the regime of the slow motional spin label by high EPRfields has showed that jump diffusion mainly affects the linewidths at the same motional rates.20 In our calculations, theintermediate jump diffusion model was considered, withcoefficients values ofR ) 5.4 × 10-10 s andâ ) -1.36.21

In Figure 8 are plotted the average of the correlation timefor differents values of the pH. As shown in the figure, thepH influences the rotational correlation time of Tempyo withrespect to all these proteins. In the acid pH range, the NH2

groups of the label molecule as well as those of the aminoacids residues are protonated. The fact thatτ shows greatervalues in this range followed by a significant decrease inthe basic pH range indicates a low mobility of spin label inacid environment, whereas an increasing of mobility can benoticed in the basic pH range. From the pH dependence ofcorrelation time (involving the mobility of the label as well),we assume that in an acid environment the mobilities of spinlabel molecules are reduced because if the formation of thehydrogen bonds between the NH2 group of the spin labeland the side chains of neighboring amino acids. In the caseof BSA, one can correlate this observation with the fact that

Table 1. Magnetic Parameters Values of the Tempyo Spin Label in Lyophilized Bovine Serum Albumin at Various pH Values

pH gxx gyy gzz Axx(G) Ayy(G) Azz(G)

11 2.0103 ( 4.3 × 10-4 2.0084 ( 4.3 × 10-4 2.0044 ( 4.3 × 10-4 4.7 ( 0.2 8.5 ( 0.4 35.5 ( 16.7 2.0011 ( 4.3 × 10-4 2.0074 ( 4.3 × 10-4 2.0050 ( 4.3 × 10-4 6.2 ( 0.3 6.6 ( 0.3 33.2 ( 0.92.5 2.0124 ( 4.3 × 10-4 2.0066 ( 4.3 × 10-4 2.0054 ( 4.3 × 10-4 8.3 ( 0.4 5.3 ( 0.3 36.0 ( 1.1

Table 2. Magnetic Parameters Values of the Tempyo Spin Label in Lyophilized Ovalbumin at Various pH Values

pH gxx gyy gzz Axx(G) Ayy(G) Azz(G)

11 2.0111 ( 4.1 × 10-4 2.0069 ( 4.1 × 10-4 2.0041 ( 4.1 × 10-4 4.3 ( 0.2 7.7 ( 0.3 34.1 ( 0.96.7 2.0012 ( 4.1 × 10-4 2.0063 ( 4.1 × 10-4 2.0053 ( 4.1 × 10-4 7.3 ( 0.3 7.2 ( 0.2 32.8 ( 0.82.5 2.0014 ( 4.1 × 10-4 2.0098 ( 4.1 × 10-4 2.0055 ( 4.1 × 10-4 4.6 ( 0.2 9.7 ( 0.4 35.6 ( 1.1

Figure 6. Experimental EPR spectrum and its subspectra of theTempyo spin label in lyophilized hemoglobin at pH ) 2.5.

Figure 7. experimental EPR spectrum and its subspectra of Tempyospin label in lyophilized hemoglobin at pH ) 11.

Rotational Correlation Times of Nitroxide Radical Biomacromolecules, Vol. 4, No. 6, 2003 1633

serum albumin undergoes reversible isomerization in the pHrange 2.7-7 from the expanded form characterized by 35%R-helix content to normal the form characterized by 55%R-helix content accompanied by a decrease inâ-sheet.22,23

It is well-known that theâ-sheet conformation favors theformation of hydrogen bonding.

By comparing the results in Figure 8, we can notice thatthe mobility of Tempyo is greater with respect to that of theheme proteins, which is not surprising if we take into accountthat hydrogen bonding opportunities depend on theâ-sheetcontent: in hemoglobin, theâ-sheets represent 50%, whereasin BSA, the percentage varies from 70% to 45%, dependingon pH. On averrage,τcyto < τHb < τovalb < τBSA.

As shown in Figure 8, the mobility of Tempyo increasesin an acid environment, followed by a slow decrease. Wesuggest that in the basic pH range, where the label is notsubject to strong electrostatic interactions, dipolar and spin-spin interactions are manifested almost with the samecontribution in the brodening of the spectrum.

Conclusions

EPR spectroscopy is very useful to study the mobility ofnitroxide radicals with respect to heme or nonheme proteinsin different environmental conditions. Noncovalent labeling

of proteins can give valuable information on the magneticinteractions between the label molecule and the paramagneticcenter of the proteins. The relevance of this interaction canbe obtained from line shape analysis: computer simulationsfor a nonheme protein assume a Gaussian line shape, whereasfor a heme protein, a weighted sum of Lorentzian andGaussian components is assumed. The contribution of theeach line shape to experimental spectrum depends on thepH. We can conclude that, on averrage,τcyto < τHb < τovalb

< τBSA.

References and Notes

(1) Morrisett, J. D.; Wien, R. W.; McConnell, H. M. The use of spinlabels for measuring distances in biological systems.Ann. N.Y. Acad.Sci. 1973, 222, 149-162.

(2) Marsh, D. InSpectroscopy and Dynamics of Molecular BiologicalSystems; Bayley, P. M., Dale, R. E., Eds.; Academic Press: London,1985; pp 209-238.

(3) Jost, P.; Griffith, O. H. Electron spin resonance and the spin labelingmethod. InMethods in Pharmacology; Chignell C., Ed.; Appleton:New York, 1972.

(4) Morrisett, J. D.; Pownall, H. J.; Gotto, A. M. Bovine serum albumin,Study of the fatty acid and steroid binding sites using spin labeledlipids. J. Biol. Chem.1975, 250, 2487-2494.

(5) Morrisett, J. D. Spin labeled enzymes. InSpin Labeling-Theory andApplication; Berliner, J., Ed.; Academic Press: New York, 1975.

(6) Frajer, P. Electron Spin Resonance Spectroscopy Labeling in Peptideand Protein Analysis. InEncyclopedia of Analytical Chemistry;Meyers, R. A., Ed.; John Wiley & Sons Ltd.: New York, 2000).

(7) Biswas, R.; Kuhne, H.; Brudvig, G. W.; Gopalan, V. Use of EPRspectroscopy to study macromolecular structure and function.Sci.Prog. 2001, 84 (1), 45-68.

(8) Hwang, J. S.; Mason, R. P.; Hwang, L.-P.; Freed, J. H. ElectronSpin Resonance Studies of Anisotropic Rotational Reorientation andSlow Tumbling in Liquid and Frozen Media. III. Perdeuterated2,2,6,6,-Tetramethyl-4-piperidoneN-Oxide and an Analysis of Fluc-tuating Torques.J. Phys. Chem.1975, 79, 489-511.

(9) Meirovitch, E.; Igner, D.; Moro, G.; Freed, J. H. Electron-spinrelaxation and ordering in smectic and supercooled nematic liquidcrystals.J. Chem. Phys.1982, 77, 3915-3938.

(10) Tanaka, H.; Freed, J. H. Electron spin resonance studies on orderingand rotational diffusion in oriented phosphatidylcholine multilayers:evidence for a new chain-ordering transition.J. Phys. Chem.1984,88, 6633-6643.

(11) Marsh, D.; Horvath, L. I. Spin label studies of structure and dynamicsof lipide and proteins in membranes. InAdVance EPR-Applicationin Biology and Biochemistry; Hoff, A. J., Ed.; Elsevier: Amsterdam,1989.

(12) Schreier, S.; Polnaszek, C. F.; Smith, I. C. P. Spin labels inmembranes.Biochim. Biophys. Acta1978, 515, 375-436.

Table 3. Magnetic Parameters Values of the Tempyo Spin Label in Lyophilized Hemoglobin at Various pH Valuesa

pH gxx gyy gzz Axx(G) Ayy(G) Azz(G)

11 2.0124 ( 4.3 × 10-4 2.0077 ( 4.2 × 10-4 2.0037 ( 4.2 × 10-4 4.8 ( 0.2 8.7 ( 0.4 34.0 ( 1 (L)

2.0112 ( 4.3 × 10-4 2.0050 ( 4.2 × 10-4 2.0061 ( 4.2 × 10-4 6.4 ( 0.3 7.8 ( 0.7 33.2 ( 0.9 (G)

6.7 2.0011 ( 4.2 × 10-4 2.0077 ( 4.2 × 10-4 2.0042 ( 4.2 × 10-4 3.3 ( 0.2 8.2 ( 0.4 33.7 ( 0.9 (L)

2.0122 ( 4.2 × 10-4 2.0048 ( 4.2 × 10-4 2.0056 ( 4.2 × 10-4 7.4 ( 0.3 8.9 ( 0.4 35.8 ( 1.1 (G)

2.5 2.0127 ( 4.2 × 10-4 2.0082 ( 4.2 × 10-4 2.0041 ( 4.2 × 10-4 4.1 ( 0.2 5.4 ( 0.2 35.6 ( 1.1 (L)

2.0117 ( 4.2 × 10-4 2.0029 ( 4.2 × 10-4 2.0051 ( 4.2 × 10-4 7.1 ( 0.3 9.4 ( 0.4 36.5 ( 1.1 (G)

a (L), Lorentzian line shape; (G), Gaussian line shape.

Table 4. Magnetic Parameters Values of the Tempyo Spin Label in Lyophilized Cytochrome c at Various pH Valuesa

pH gxx gyy gzz Axx(G) Ayy(G) Azz(G)

11 2.0155 ( 4.2 × 10-4 2.0109 ( 4.2 × 10-4 2.0056 ( 4.2 × 10-4 4.8 ( 0.2 8.7 ( 0.4 33.0 ( 0.9 (L)

2.0124 ( 4.2 × 10-4 2.0140 ( 4.2 × 10-4 2.0087 ( 4.2 × 10-4 6.4 ( 0.3 7.8 ( 0.3 34.2 ( 0.9 (G)

6.7 2.0141 ( 4.2 × 10-4 2.0118 ( 4.2 × 10-4 2.0049 ( 4.2 × 10-4 6.0 ( 0.3 5.9 ( 0.3 33.0 ( 0.9(L)

2.0136 ( 4.2 × 10-4 2.0113 ( 4.2 × 10-4 2.0057 ( 4.2 × 10-4 5.2 ( 0.2 3.7 ( 0.2 35.3 ( 1 (G)

2.5 2.0183 ( 4.2 × 10-4 2.0092 ( 4.2 × 10-4 2.0025 ( 4.2 × 10-4 3.3 ( 0.2 7.1 ( 0.4 32.1 ( 0.8 (L)

2.0124 ( 4.2 × 10-4 2.0129 ( 4.2 × 10-4 2.0087 ( 4.2 × 10-4 7.8 ( 0.4 6.4 ( 0.3 33.4 ( 0.9 (G)

a (L), Lorentzian line shape; (G), Gaussian line shape.

Figure 8. Correlation times (τ) as a function of pH for Tempyo spinlabel in lyophilized cytochrome c (9), hemoglobin (b), ovalbumin (2),and bovine serum albumin (1).

1634 Biomacromolecules, Vol. 4, No. 6, 2003 Cavalu and Damian

(13) Goldman, S. A.; Bruno, G. V.; Polnaszek, C. F.; Freed, J. H. AnESR study of anisotropic rotational reorientation and slow tumblingin liquid and frozen media.J. Chem. Phys. 1972, 56, 716-735.

(14) Poole, C. P., Jr.; Farach, H. A. InTheory of Magnetic Resonance;John Wiley & Sons: New York, 1987; pp 319-321.

(15) Schneider, D. J.; Freed, J. H. InSpin Labeling Theory andApplications; Berliner, L. J., Reuben, J., Eds.; Plenum Press: NewYork, 1989; pp 1-76.

(16) Marsh, D. InSpin Labeling Theory and Applications; Berliner, L.J., Reuben, J., Eds.; Plenum Press: New York, 1989; pp 255-303.

(17) Berliner, L. J.Spin Labeling, Theory and Application; AdademicPress: New York, 1976.

(18) Budker, V.; Du, J.-L.; Seiter, M.; Eaton, G. R.; Eaton, S. S.;Electron-electron spin-spin interaction in spin labeled low-spinmethemoglobin.Biophys. J.1995, 68, 2531-2542.

(19) Rakowsky, M. H.; More, M. K.; Kulikov, A. V.; Eaton, G. R.; Eaton,S. S. Time-Domain Electron Paramagnetic Resonance as a Probe ofElectron-Electron Spin-Spin Interaction in Spin-Labeled Low-SpinIron Porphyrins.J. Am. Chem. Soc. 1995, 117, 2049-2057.

(20) Earle, K. A.; Budil, D. E.; Freed, J. H. 250-GHz EPR of Nitroxidein the Slow-Motional Regime: Models of Rotational Diffusion.J.Phys. Chem. 1993, 97, 13289-13297.

(21) Eaton, G. R.; Eaton, S. S. Interaction of spin labels with transitionmetals.Coord. Chem. ReV. 1978, 26, 207-262.

(22) Foster, J. F. InAlbumin Structure, Function and Uses; Rosenoer, V.M., Oratz, M., Rothschild, M. A., Eds.; Pergamon: Oxford, 1977;pp 53-84.

(23) Carter, D. C.; Ho, J. X. Structure of Bovine Serum Albumine.AdV.Protein Chem.1994, 45, 153-203.

BM034093Z

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