Origin of the Temperature Dependent Isotropic Hyperfine Coupling of

Devkumar Mustafi, Alejandro Sosa-Peinado, Vanita Gupta, David J. Gordon, and Marvin W. Makinen. Biochemistry 2002 41 (3), 797-808. Abstract | Full Tex...
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J. Phys. Chem. 1995, 99, 11370- 11375

11370

Origin of the Temperature Dependent Isotropic Hyperfine Coupling of the Vinylic Proton of Oxypyrrolinyl Nitroxyl Spin-Labels Devkumar Mustafi* Department of Biochemistry and Molecular Biology, The University of Chicago, Cummings Life Science Center, 920 East 58th Street, Chicago, Illinois 60637

Heikki Joela Department of Chemistry, University of Jyvaskyla, SF-40500 Jyvaskyla, Finland Received: March 20, 1995; In Final Form: May 15, 1995@

Proton ENDOR spectra of the spin-label 2,2,5,5-tetramethyl-l-oxypyrroline-3-carboxylic acid in methanol, chlorofodtoluene, and toluene exhibited well resolved features, permitting accurate estimation of hyperfine coupling (hfc) components of the vinylic and methyl protons. The magnitude and relative sign of the isotropic hfc Aiso for these two classes of protons were determined by electron nuclear double resonance (ENDOR) and TRIPLE spectroscopy. While the isotropic hfc of methyl protons was temperature independent, Aiso of the vinylic proton was temperature dependent in fluid and frozen solutions. The temperature dependence was not influenced by solvent polarity, but at T I 50 K, Aiso of the vinylic proton became temperature independent. The temperature dependence is explained on the basis of the out-of-plane vibration of the vinylic proton. Above 50 K, the out-of-plane vibration of the C-HV bond causes the vinylic proton to become displaced from the nodal plane. The ratio of the measured Aiso values of the vinylic proton and deuteron indicated that the vibrational mechanism is responsible for the temperature dependent isotropic hfc of the vinyl proton of the oxypyrrolinyl nitroxyl spin-labels.

Introduction Nitroxyl spin-labels are a remarkable series of free radicals, interest in which has grown as a consequence of their extensive use in biological studies.' The spin-label probe 2,2,5,5tetramethyl- 1-oxypyrroline, in particular, has been extremely useful in monitoring the concentration of dissolved oxygen in biological system^.^-^ These studies depend on precise measurements of the electron paramagnetic resonance (EPR5) line width and the isotropic hyperfine coupling of the vinylic proton in the oxypyrrolinylring. Thus far, the superhyperfine couplings have usually been determined by EPR method^.^-^ However, EPR is not very sensitive to small hyperfine couplings. In contrast, by using electron nuclear double resonance (ENDOR5) techniques on spin-label probes in frozen solution, we have obtained very accurate estimates of both the isotropic and dipolar hfc components for protons in the oxypyrrolinyl ring and in the side chains of spin-labeled derivatives6 For spin-label EPR oximetry, the vinylic proton is used as a spectroscopic monitor, and its Aiso is temperature dependent, in contrast to other spin-label proton^.^ Here we investigate the temperature dependence of the isotropic hf couplings of the vinylic proton by ENDOR and TRIPLE spectroscopy. The value of Aiso of the vinylic proton has been determined over the temperature range 5-340 K by ENDOR, and its sign relative to that of the methyl protons and the nitroxyl nitrogen was obtained from TRIPLE resonance spectra. In ENDOR spectra of spin-labels in fluid and frozen solutions of toluene, chloroform/ toluene, and methanol, we have also observed a small solvent dependence of Aiso of the vinyl proton. The small solvent dependence of Aiso is probably due to the polarity of solvents, which causes a polarization of the n-electron charge and the spin density distribution of the nitrogen and oxygen atoms of

* Author

to whom correspondence should be addressed. Telephone:

(312) 702-1667. Fax: (312) 702-0439. E-mail: [email protected]. @Abstractpublished in Advance ACS Abstracts, June 15, 1995.

0022-365419512099- 11370$09.00/0

the nitroxyl group. However, the temperature dependence was not affected by solvent polarity and is explained on the basis of an out-of-plane vibration of the vinylic proton. From analysis of ENDOR-determined values of Aiso of protons and deuterons in spin-labels, we have shown that the vibrational mechanism is responsible for the temperature dependent isotropic hyperfine coupling of the vinyl proton in spin-labels.

Experimental Procedures General Materials. The spin-label 2,2,5,5-tetramethyl-loxypyrroline-3-carboxylic acid was obtained by hydrolysis of the corresponding carboxamide (Aldrich Chemical Co., Inc., Milwaukee, WI 53233) by the method of Rozant~ev.~The [2HI &pin-label 2,2,5,5-[2H~ 21tetramethyl- 1-0xy-4-[~HIpyrroline3-carboxylic acid was synthesized, as descxibed by us previously.6b The [2H,&pin-label 2,2,5,5-[2H,zltetramethyl-l-oxypyrroline3-carboxamide was obtained from Dr. Howard J. Halpem of the Department of Radiation and Cellular Oncology at the University of Chicago.4a MBBA5 was obtained from Aldrich. The deuterated solvents [2H4]methanol(99.8%), [*H]chloroform (99.8%), and [2H8]toluene (99.6%) were obtained from'cambridge Isotope Laboratories, Inc. (Wobum, MA 01801). EPR and ENDOR. EPR and ENDOR spectra of the spinlabel in frozen solutions were recorded with an X-band Bruker ER200D spectrometer equipped with an Oxford Instruments ESRlO liquid helium cryostat, as previously described.8 Typical experimental conditions for ENDOR measurements in frozen solutions were as follows: sample concentration, 4 x M; sample temperature, 5-100 K; microwave frequency, 9.46 GHz; incident microwave power, 1.28 mW; rfpower, 50 W with 12.5 kHz frequency modulation; and 10 kHz modulation depth of the rf field. ENDOR and TRIPLE spectra of the spin-label in fluid solutions were recorded with a Bruker ER200 D-SRC spectrometer, as previously de~cribed.~ For measurements in fluid solutions of perdeuterated solvents and in MBBA,'O a spinlabel concentration of 1 x M was used, and the samples

0 1995 American Chemical Society

Temperature Dependent Isotropic Hyperfine Coupling

J. Phys. Chem., Vol. 99, No. 29, I995 11371

Figure 2. Temperature dependence of the isotropic hyperfine couplings of the vinylic proton of 2,2,5,5-tetramethyl- 1-oxypyrroline-3-carboxylic

, 8.0

-0.5

0

0.5

1.0

MHz

Figure 1. ENDOR and TRIPLE resonance spectra of spin-label 2,2,5,5tetramethyl-1-oxypyrroline-3-carboxylicacid in a fluid solution of [%4]methanol. In the top panel, the top and bottom ENDOR spectra were recorded with sample temperatures of 183 and 226 K, respectively. The ENDOR splittings for the vinylic and methyl protons of the spinlabel are indicated in a stick diagram by aHVand aCH3,respectively. In the bottom panel, the general TRIPLE resonance spectrum was recorded at 183 K and the high-frequency transition of the vinylic proton indicated by the arrow was pumped. The abscissa indicates the ENDOR shift (measured ENDOR frequency minus free nuclear Larmor frequency).

were degassed to Torr through alternate freezehhaw cycles. Typical experimental conditions were as follows: sample temperature, from -230 K to the freezing point of the respective solvents; microwave frequency, 9.47 GHz; incident microwave power, 50 mW; rf power, 300 W for ENDOR and 400 W for TRIPLE; 60 kHz modulation depth of the rf field for 'H-ENDOR; and 30 kHz modulation depth of the rf field for 2H-ENDOR.

Results A. Proton ENDOR and TRIPLE Spectra of 2,2,5,5Tetramethyl-1-oxypyrroline-3-carboxylicAcid. Figure 1 illustrates the proton ENDOR and TRIPLE resonance spectra of 2,2,5,5tetramethyl- 1-oxypyrroline-3-carboxylicacid in a fluid solution of [*&]methanol. The top panel shows two sets of ENDOR features that are symmetric about the free proton frequency of 13.98 MHz and are labeled as the isotropic hfc components for vinylic and methyl protons. As we increase the sample temperature, the ENDOR splitting of the vinylic proton decreases, while the ENDOR splitting for methyl protons remains unchanged. The only temperature variation in ENDOR absorption features of methyl protons is the line width, as we have observed for other methyl groups.6b Two representative spectra are shown in Figure 1 in which the decreased line splitting for the vinyl proton at higher temperatures is indicated by dashed lines in the bottom ENDOR spectrum. A similar observation was reported by Hyde and Subczynski on the basis of EPR studies of the spin-labeled carboxamide in water in the temperature range from 1 to 80 oC.3b The bottom panel of Figure 1 shows the TRIPLE resonance spectrum of the spin-label. In the general TRIPLE experiment,

acid. Data obtained for the spin-label in perdeuterated methanol, chlorofodtoluene (50:50 v/v), and toluene are shown in circles, triangles, and squares, respectively. The open and closed symbols represent data obtained in fluid and frozen solutions, respectively. The value of A,,, of -1.46 MHz in fluid toluene solution at 185 K, as indicated by the half-filled diamond symbol, was taken from Kirste et al.I3 Dashed lines are drawn through the experimentally measured points.

transitions of different nuclei are driven simultaneously.' One ENDOR transition is irradiated with saturating rf power at constant frequency. The entire ENDOR range is then swept to obtain the TRIPLE resonance spectrum. If other protons (or nuclei) belong to the same paramagnetic molecule, the TRIPLE resonance spectrum exhibits redistribution of intensities relative to the ENDOR spectrum. In the spin label, both methyl and vinylic protons are associated with the oxypyrroline ring. When the high-frequency ENDOR transition of the vinylic proton is pumped, the intensity of the corresponding low-frequency transition increases, as illustrated in the bottom panel of Figure 1. A similar pattern of intensity change, increased peak-topeak amplitude of the low-frequency transition and decreased signal amplitude of the high-frequency transition, is also observed for the methyl protons. This is shown graphically by the lines connecting the high-frequency peaks to the lowfrequency peaks of the vinylic and methyl protons, which slope in the same direction. Thus, the TRIPLE experiments demonstrate that the sign of the isotropic hfc components for methyl and vinylic protons is the same. On the basis of NMR studies of a series of aliphatic nitroxide radicals, it has been shown that the sign of Ai,, for methyl protons is negative.'* Therefore, the sign of Ai,, for both the vinylic and methyl protons is assumed to be negative. B. Temperature Dependence of the Proton Isotropic hfc Components. Figure 2 illustrates the temperature dependence of the isotropic hf coupling of the vinylic proton of the spinlabel in three different solvent systems. The values of Ai,, in fluid solution were obtained directly from ENDOR spectra. For frozen solutions, however, the values of Aiso were estimated from the experimentally determined parallel and perpendicular hfc components using the relation (AII 2A1)/3 = Aiso. For ENDOR, sample concentrations of 4 x and 1 x M in frozen solutions and in fluid solutions, respectively, were used. For ENDOR in frozen solution, higher sample concentrations were used to improve the signal-to-noise ratio of resonance absorption features. However, in frozen solutions the ENDORdetermined values of the All and A 1 components, from which the value of Aiso was estimated, were found to be identical for

+

11372 J. Phys. Chem., Vol. 99, No. 29, 1995

Mustafi and Joela

samples in the concentration range from 4 x low4to 4 x M, indicating no concentration dependence. The value of A,,, decreases with decreasing temperature and finally reaches a plateau below 50 K. As seen in Figure 2, the values of A,,, in the temperature independent region obtained in three solvent systems are slightly different, indicating that the magnitude of A,,, for the vinylic proton is also solvent dependent. However, the three plots in Figure 2 exhibit almost identical slopes, indicating that the temperature dependence of A,,, for the vinylic proton is not affected by solvent polarity. On the other hand, a value for A,,, of -0.66 f 0.03 MHz for the methyl protons was found in the temperature range -180235 K in fluid solutions of methanol, chlorofodtoluene, and toluene. An identical value of A,,, has been reported by Kirste er al. for the methyl protons of the same spin-label in toluene at 185 K.I3 In addition to the values of A,,, for the vinylic proton of spinlabels in three solvent systems as reported here and as seen in 1 I I I I -100 -50 0 50 100 Figure 2, we have also obtained its isotropic hf coupling in the temperature range 120-340 K using spin-labels in MBBA.Io kHz Under isotropic conditions, the values of A,,, of -1.19 MHz at Figure 3. ENDOR spectra of 2,2,5,5-[2H12]tetramethyl1-0xy-4-[~H]330 K and - 1.17 MHz at 340 K for HVwere obtained directly pyrroline-3-carboxylic acid in a fluid solution of methanol at 200 K. The ENDOR splittings for the vinylic and methyl deuterons are from the ENDOR spectra. These hf couplings for HV are and aCD3,respectively.The abscissa indicated in a stick diagram by comparable to those that were reported by Hyde and Subczynski indicates the ENDOR shift, symmetric about the free deuterium for spin-labels in water in the temperature range 274-353 K.3b frequency of 2.187 MHz. In anisotropic solutions of MBBA, we have estimated A,,, for HVfrom ENDOR-determined All and A1 hfc components in the tion of only two pairs of resonance features for each proton in temperature range 120-240 K. The temperature dependence spin-labels requires that the observed ENDOR splittings corof A,,, for HVof spin-labels in MBBA over the range 120-340 respond to axially symmetric principal hfc components. The K is found to be linear and is similar to those that are shown in principal hfc components, as obtained from ENDOR spectrosFigure 2. Moreover, since MBBA is an anisotropic frozen copy, can be expressed in terms of dipolar and isotropic consolution below 288 K, at relatively higher temperatures (120tributions: All = AllD Ais, and A1 = AID Ai,,. Since the 240 K), the dipolar hfc components and the dipolar distance to trace of the dipolar hfc components of the hf tensor is zero (Le., the vinylic proton can be estimated from ENDOR-determined AllD 2AlD = 0), the isotropic contribution can be extracted hfc components. directly from ENDOR-determined principal hfc components as C. Deuterium Isotropic hfc Components. We have also Ais, = (All 2A1)/3. In earlier ENDOR studies of spin-labeled measured isotropic hf couplings of deuterons substituted at the derivatives, we have demonstrated the validity of this equation.6 vinyl and methyl carbon atoms of spin-labels. Values of A,,, Using the calculated value of Ai,, to estimate dipolar hfc for the vinylic and methyl deuterons were obtained by ENDOR components, we have shown that, even for protons located in spectroscopy for samples in fluid solutions of natural abundance the immediate vicinity of the nitroxyl group, like the vinylic and perdeuterated methanol. Figure 3 illustrates the deuterium and two amide protons of spin-labeled carboxamide, the ENDOR spectrum of 2,2,5,5-[*H1~]tetramethyl-l-oxy-4-[~H]-ENDOR-determined electron-proton dipolar distances are in pyrroline-3-carboxylic acid in methanol. By comparing the excellent agreement with distances calculated on the basis of ENDOR spectra of [*Ho], [2H12],and [2H~&pin-labelsin fluid X-ray-defined atomic coordinate^.^^-'^ solutions of methanol, two sets of resonance features, symmetric The isotropic hyperfine structure of a free radical is related about the free deuterium frequency of 2.2 MHz, were assigned to the spin density of the unpaired electron at the positions of as the isotropic hfc components for the vinylic and methyl the magnetic nuclei in the molecule. In the 2,2,5,5-tetramethyldeuterons, as seen in Figure 3. The magnitudes of A,,, of 0.182 1-oxypyrrolinyl spin-label probe, the spin density of the unpaired f 0.008 and 0.102 f 0.008 MHz for the vinylic and methyl electron is confined almost entirely to the nitrogen and oxygen deuterons, respectively, were measured at 200 K. Because of atoms of the nitroxyl group.I4,l5 However, the observed the poor signal-to-noise ratio at the lower frequency region of isotropic hyperfine splittings for the methyl and vinylic protons 1.6-2.6 MHz, *H-ENDOR measurements were carried out only of spin-labels indicate that a small fraction of unpaired spin over a small temperature range. The magnitude of A,,, for the density is also present in the ring carbons, particularly at the vinylic deuteron changes from 0.182 MHz at 200 K to 0.178 vinyl carbons. McConnell has shown that the isotropic proton MHz at 210 K, while for the methyl deuterons the value of A,,, hyperfine splitting A,,, is related to the unpaired n-electron spin remains the same. density e at the carbon atom by the simple equation

+

+

+

+

Discussion A. Estimation of the Unpaired Electron Spin Density from Aiso. In fluid solutions, the anisotropic hfc contributions average out, and the value of Ai,, is obtained directly from ENDOR splittings. In frozen solutions, however, the hyperfine interaction includes primarily the electron-nuclear dipolar interaction AD and to a first-order approximation the isotropic Fermi contact interaction Ai,,. In frozen solutions, the observa-

where Q is a semiempirical constant.I6 Using the value of Q = -69 MHz,16 we estimate the spin density at the carbon atom on the basis of the isotropic hf splitting of the proton which is a-bonded to the carbon atom. The fractional spin density of 2.6% for the vinyl carbon was calculated from the lowtemperature plateau value of Aiso for the vinylic proton.

J. Phys. Chem., Vol. 99, No. 29, 1995 11373

Temperature Dependent Isotropic Hyperfine Coupling

B. Small Solvent Dependence of Ai, of the Vinylic Proton. The temperature invariant values, - 1.76 and - 1.81 MHz, of Aiso for the vinylic proton of the spin-label in toluene and methanol, respectively, indicate that Aiso is also solvent dependent. The small solvent dependence of Aiso is probably due to radical-solvent interactions which cause polarization of the n-electron charge and spin density distribution of the nitrogen and oxygen atoms of the nitroxyl group. Therefore, the hyperfine splittings of I4N vary with the strength and nature of the radical-solvent complexes. In this study the values of Aiso for the nitroxyl I4N were found to be 14.30 G in toluene, 14.80 G in chlorofodtoluene, and 14.96 G in methan01.I~ Similarly, for di-tert-butyl nitroxide, Griffith et al. report that the isotropic hf splittings for I4N also increase as solvent polarity increases.'* In aprotic solvents, much of the unpaired electron is associated with the oxygen atom. Solvents such as water or methanol may interact with the oxygen atom of the nitroxyl group by hydrogen bonding. The dipolar forces from these polar solvent molecules increase the electron affinity of the oxygen atom. This stabilizes the N-0 three-n-electron distribution in which the paired electrons (lone pair) are located on the oxygen atom and the unpaired electron is located on the nitrogen, which in turn increases the I4N hf coupling. A similar solvent dependent phenomenon is found in quinone radi~a1s.I~Small fractional spin densities at the carbon atoms in the oxypyrrolinyl ring arise from spin delocalization. As the fractional spin density on the nitroxyl nitrogen increases, a slight enhancement of fractional spin density should occur at the ring carbons. The relatively higher spin density in the vinyl carbon must increase the magnitude of Ai,, for the vinylic proton of the spin-label in a polar solvent, as illustrated in Figure 2. However, the results clearly show that the intrinsic temperature dependence of Aiso for the vinylic proton of the spin-label is independent of solvent polarity. C. Origin of the Temperature Dependence of Aiso of the Vinylic Proton in Spin-Labels. The isotropic hf coupling of a proton is observed if the wave function of the unpaired electron has a finite amplitude at the 1s orbital of that proton. The strength of the isotropic hf interaction can be affected by perturbation of the molecular geometry of radicals. The most likely mechanism of the temperature dependence would be an out-of-plane vibration of atoms with respect to the pyrrolinyl ring. Figure 4 illustrates the root mean square (r.m.s.) fluctuations of positions for all atoms of the spin-label molecule calculated on the basis of isotropic temperature factors determined from X-ray diffraction studies of 2,2,5,5-tetramethyl- 1oxypyrroline-3-carboxamide.20We have also calculated the r.m.s. fluctuations of positions for all nonhydrogen atoms of the spin-labeled carboxylic acid from isotropic temperature factors determined from X-ray diffraction studies of 2,2,5,5tetramethyl-1-oxypyrroline-3-carboxylicacid.21 The r.m.s. fluctuations of positions for all non-hydrogen atoms in the spinlabel carboxamide are identical to those that were calculated for the corresponding acid. In the latter studies, isotropic temperature factors for hydrogen atoms are not given and, therefore, cannot be compared. For this unsaturated spin-label, the oxypyrrolinyl ring has a planar configuration. The r.m.s. fluctuations of positions for the four ring carbons and the nitroxyl nitrogen are equivalent. The fluctuation of the atomic position of the nitroxyl oxygen is slightly higher than that of the other atoms in the ring. From the EPR studies we have estimated values of Aiso of 14.96 G at 298 K and 15.11 G at 40 K for the nitroxyl I4N of the spin-label in methanol. Reddoch and Konishi reported a similar increase of Aiso of -0.1 G by lowering the temperature from 300 to 173 K for nitroxide

R

H"

0.6

z

0'

o.*w NI 01 C l

c)C4

H"C5 C6

Atom Number

Figure 4. Illustration of the atomic numbering scheme and the r.m.s. fluctuations of positions for a l l atoms of the 2,2,5,5-tetramethyl-loxypyrroline moiety. In the atomic numbering scheme the label R is used to designate the carbon atom of the carboxamide or carboxylic acid group.

TABLE 1: Temperature Dependence of the Hyperfine Coupling Component@ and the Electron-Proton Distanceb of the Vinylic Proton in the 2,2,5,5-Tetramethyl-l-ox~p~rrolinyl Spin-Label T W

Ail

Al

A,,,

Alp

4.5 10.0 19.5 39.5 59.5 80.0 100.0

1.148 1.148 1.148 1.148 1.165 1.183 1.216

3.295 3.295 3.295 3.288 3.262 3.240 3.206

In Methanol -1.814 -1.814 -1.814 -1.809 -1.786 -1.766 -1.732

120.0 150.0 170.0 220.0

1.32 1.34 1.37 1.45

3.08 3.04 2.97 2.84

In MBBA -1.61 -1.58 -1.52 -1.41

AID

r(A)

2.962 2.962 2.962 2.957 2.951 2.949 2.948

-1.481 -1.481 -1.481 -1.479 -1.476 -1.474 -1.474

3.77 3.77 3.77 3.77 3.77 3.77 3.77

2.93 2.92 2.89 2.86

-1.47 -1.46 -1.45 -1.43

3.78 3.79 3.80 3.81

hfc components are given in units of MHz. The hfc values of HV for the sample in frozen solution are obtained at the temperature range 4.5-100 K, whereas, for the sample in MBBA,'O they are obtained at 120 K and above. According to the constraint All > 0 > A l , the sign of AL is considered to be negative. Estimated error in r of 0.01 -0.02

8, at 4.5-100 K and of 0.02-0.04 8, at 120-220 K is based on an uncertainty in frequency of 0.01-0.03 MHz due to the ENDOR line width.

radicals in ethanol.22 This small change in I4N isotropic coupling has a negligible effect on overall spin density redistribution between the nitroxyl nitrogen and oxygen atoms. In fact, as seen in Table 1, the values of the dipolar hfc components, estimated from the ENDOR-determined parallel and perpendicular hfc components in the temperature range 5-220 K, show no temperature dependence. Therefore, the ENDOR-determined electron-proton dipolar distance for the vinylic proton remains constant over the temperature range, as listed in Table 1. The temperature dependent values of the observed hfc components All and A l , thus, reflect changes only in the value of Aiso. Figure 4 shows that the r.m.s. fluctuation of the atomic position of the vinylic proton is much higher than the fluctuations of all atoms in the oxypyrrolinyl ring and of those that

11374 J. Phys. Chem., Vol. 99, No. 29, 1995

t Figure 5. Valence orbitals of the vinyl carbons C3 and C4 of the oxypyrrolinyl ring of the spin-label. The dashed lines through atoms and occupied orbitals represent the plane of the oxypyrrolinyl ring. The 2p, orbitals of the vinyl carbons are perpendicular to the molecular plane of the oxypyrrolinyl ring.

TABLE 2: Comparison of Calculated and ENDOR-Determined Values of A h (in MHz) of the Vinylic and Methyl Protons in the Tetramethyl-1-oxypyrrolinyl Spin-Label A,,, (ENDOR)b

A,,, (calcY

proton

I'

IId

vinyl methyl

-1.16 -0.66

-1.58 -0.91

- 1.52 ( + O M ) -0.66 (f0.03)

Values of A,,, were calculated using eqs 3 and 4 and on the basis of ENDOR-determined isotropic hf couplings of 0.178 and 0.102 MHz for deuterons at the vinyl and methyl positions, respectively, for samples in a fluid solution of methanol at 210 K. ENDOR-determined values of A,,, for the vinyl and methyl protons were also obtained for samples in a fluid solution of methanol at 210 K. Using eq 3. Using eq 4.

are directly bonded to the ring, including the carbon atom of the carboxamide group. A maximum deviation of 24' for the vinylic C-H bond with respect to the mean plane of the oxypyrrolinyl ring was estimated from the r.m.s. fluctuations. Figure 5 illustrates the valence orbitals of the C3 and C4 vinyl carbons and the vinylic proton. The dashed lines represent the plane of the five-membered ring. The fractional spin density of 0.026 for the vinyl carbon, obtained from the low-temperature plateau value of A,,, for the vinylic proton of the spin-label in methanol, is localized in the 2pz atomic orbital. The negative isotropic hf coupling demonstrates that the spin density on the vinylic proton is negative, and it must be opposite in sign to the spin density on the carbon atom, because the spin on the proton tends to be antiparallel to the spin on the carbon atom due to ordinary covalent exchange bonding.23 The C(4)-HV fragment in this case is very similar to the >C-H fragment of the malonic acid radical, where the unpaired electron is in the 2pz orbital and the a-proton lies in the nodal plane.23 The large r.m.s. fluctuation of the atomic position of the vinylic proton implies that at higher temperatures the out-of-plane vibration of the C(4)-HV bond causes a large displacement of the vinylic proton from the plane of the five-membered oxypyrrolinyl ring. At very l o w temperatures (T 5 50 K) the vinylic proton lies in the nodal plane, and the spin density on the carbon atom does not contribute to spin polarization. As the temperature increases, the C-H vibrator of the vinylic group no longer lies in the nodal plane, and the positive spin density in the pz orbital of the carbon atom and negative spin density at the vinyl proton decrease. Similar results have been reported by Derbyshire for the >C-OH radical system.24 D. Vibrational Mechanism: Cause of the Temperature Dependence of Ais0 of the Vinyl Proton. The isotropic hf couplings arise from the fact that the magnetic moments of the electron and nuclei are coupled via the so-called Fermi contact interaction. The expectation value of the isotropic hf coupling of a given nucleus is given by eq 2 ,

Mustafi and Joela

where g is the spectroscopic g-factor of the electron, B is the Bohr magneton, gi is the nuclear g-factor of the ith nucleus, BN is the nuclear magneton, and Ily(i)lav2 is the density of the unpaired electron at the ith nucleus averaged over the vibrational motions.25 The calculation of the hf splitting thus requires an evaluation of lly(i)lav2.However, in the absence of vibrational motions, the splitting constant for a proton can be easily obtained from the isotropic hf constant of the corresponding deuteron a D according to eq 3, using the value of gH/gD = 6.5144. On H '

=adH/gD

(3)

the other hand, if a vibrational mechanism is responsible for the hf splitting, then a~ can be calculated according to eq 4 by

(4) taking into account the mean-square vibration amplitude term Ily(i)lav2. The latter term is inversely proportional to the square root of the reduced mass of the vibrator. Neglecting the proper normal modes, this implies that UH = 6.5144(1.3625)a~. ..We have calculated isotropic hf splitting constants for the vinylic and methyl protons by using eqs 3 and 4 and on the basis of ENDOR-determined values of Ai,, for the corresponding deuterons. The results are summarized in Table 2. The calculated value of Aiso of -0.66 MHz for the methyl protons on the basis of eq 3, using the ENDOR-determined deuterium coupling for the methyl group and the ratio of the corresponding g-factors, is in excellent agreement with the observed isotropic hf coupling of the methyl protons. On the other hand, for the vinylic proton, the calculated value of Aiso using eq 3 , as seen in Table 2 for case I, does not agree with the ENDORdetermined value of Ai,,, The observed coupling of - 1.52 MHz at 210 K for the vinylic proton corresponds closely with the calculated value for case 11, as seen in Table 2, in which case both the g and vibrational amplitude factors were taken into consideration. The results in Table 2 for the observed hf couplings of the vinylic proton and deuteron in spin-labels are best explained by slower motion of the heavier deuteron nucleus compared to the proton. The results clearly indicate that the vibrational mechanism is responsible for the temperature dependence of the isotropic hfc of the vinylic proton in spin-labels. The results also indicate that there is no temperature-induced spin density variation at the methyl carbons. The residual spin density delocalization is likely to be restricted to the oxypyrrolinyl ring. The sp3 hybridized methyl carbons are situated above and below the five-membered ring of the spin-label. The isotropic hf coupling of the methyl protons is not affected by a dynamical change of oxypyrrolinyl nitroxyl spin-labels at higher temperature.

Acknowledgment. We thank Professor M. W. Makinen for helpful discussions and support. We are also indebted to Professor H. M. McConnell for helpful discussion and his suggestion of measuring isotropic hyperfine couplings of deuterons in spin-labels. This work was supported by a grant from the National Institutes of Health (GM 21900). References and Notes (1) (a) Hamilton, C. L.; McConnell, H. M. In Strucrural Chemistry and Molecular Biology; Rich, A., Davidson, N., Eds.; W. H. Freeman: San Francisco, 1968; pp 115-149. (b) McConnell, H. M.; Gaffney-McFarland, B. Q. Rev. Biophys. 1970, 3, 91. (c) Berliner, L. J., Ed.; Spin Labeling: Theory and Application; Academic Press: New York, 1976.

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Temperature Dependent Isotropic Hyperfine Coupling (2) Sarna, T.; Duleba, A.; Korytowski, W.; Swartz, H. Arch. Biochem. Biophys. 1980, 200, 140. (3) (a) Lai, C. S.; Hopwood, L. E.: Hyde, J. S.; Lukiewicz, S. Proc. Natl. Acad. Sci. U.S.A. 1982, 79, 1166. (b) Hyde, J. S.; Subczynski, W. K. J. Magn. Reson. 1984, 56, 125. (4) (a) Halpem, H. J.; Peric, M.; Nguyen, T.-D.; Spencer, D. P.; Teicher, B. A,; Lin, Y. J.; Bowman, M. K. J. Magn. Reson. 1990,90,40. (b) Halpem, H. J.; Pou, S.; Peric, M.; Yu, C.; Barth, E.; Rosen, G. M. J. Am. Chem. SOC. 1993, 115, 218. (5) Abbreviations: ENDOR, electron nuclear double resonance; EPR, electron paramagnetic resonance; hf, hyperfine; hfc, hyperfine coupling; MBBA, N-(4-methoxybenzylidene)-4-butylaniline;r.m.s., root mean square. (6) (a) Wells, G. B; Makinen, M. W. J. Am. Chem. SOC. 1988, 110, 6343. (b) Mustafi, D.; Sachleben, J. R.; Wells, G. B.; Makinen, M. W. J. Am. Chem. SOC. 1990, 112, 2558. (c) Wells, G. B.; Mustafi, D.; Makinen, M. W. J. Am. Chem. SOC. 1990,112,2566. (d) Joela, H.; Mustafi, D.; Fair, C. C.; Makinen, M. W. J. Phys. Chem. 1991, 95, 9135. (e) Mustafi, D.; Boisvert, W. E.; Makinen, M. W. J. Am. Chem. SOC. 1993, 115, 3674. (f) Mustafi, D.; Wells, G. B.; Joela, H.; Makinen, M. W. Free Radical Res. Commun. 1990, 10, 95. (7) Rozantsev, E. G. Free Nitroxyl Radicals;Plenum Press: New York, 1970; Chapter 9, pp 205-206. (8) (a) Yim, M. B.; Makinen, M. W. J. Magn. Reson. 1986, 70, 89. (b) Mustafi, D.; Makinen, M. W. Inorg. Chem. 1988, 27, 3360. (9) Joela, H.; Kasa, S.; Miikela, R.; Salo, E.; Hannonen, K. Magn. Reson. Chem. 1990, 28, 261. (10) MBBA, N-(4-methoxybenzylidene)-4-butylaniline,is an isotropic liquid solvent at higher temperature (> 3 15 K). It forms a liquid crystalline phase in the temperature range 288-314 K. At 288 K or below, it is an anisotropic frozen solution. (1 1) Mobius, K.; Biehl, R. In Multiple Electron Resonance Spectroscopy; Dorio, M. M., Freed, J. H., Eds.; Plenum Press: New York, 1979; pp 475507.

(12) Kreilick, R. W. J. Chem. Phys. 1967, 46, 4260. (13) Kirste, B.; Kriiger, A.; Kurreck, H. J. Am. Chem. SOC. 1982, 104, 3850. (14) Mustafi, D.; Joela, H.; Makinen, M. W. J. Magn. Reson. 1991, 91, 497. (15) (a) Hayat, H.; Silver, B. L. J. Phys. Chem. 1973, 77,72. (b) Davis, T. D.; Christoffersen, R. E.; Maggiora, G. M. J. Am. Chem. SOC.1975.97, 1347. (16) (a) McConnell, H. M. J. Chem. Phys. 1956,24,632. (b) McConnell, H. M. J. Chem. Phys. 1956,24,764. (c) McConnell, H. M. J. Chem. Phys. 1958, 28, 1188. (17) The hf coupling A has the dimensions of energy. It may also be expressed as a frequency Ah.The hfc components for protons as determined by ENDOR are expressed in units of megahertz, whereas the isotropic hf couplings for the nitroxyl nitrogen as determined by EPR are expressed in gauss. Division by a factor of 2.81 converts the values in megahertz to the values in gauss. This is based on the electronic g-value of 2.0061 for oxypyrrolinyl nitroxyl spin-labels. (18) Griffith, 0. H.; Dehlinger, P. J.; Van, S. P. J. Membr. Biol. 1974, 15, 159. (19) Gendell, J.; Freed, J. H.; Fraenkel, G. K. J. Chem. Phys. 1962,37, 2832. (20) Turley, J. W.; Boer, F. P. Acta Crystallogr.,Sect. B 1972,28, 1641. (21) Boeyens, J. C. A.; Kruger, G. J. Acta Crystallogr., Sect. B 1970, 26, 668. (22) Reddoch, A. H.; Konishi, S. J. Chem. Phys. 1979, 70, 2121. (23) (a) McConnell, H. M.; Heller, C.; Cole, T.; Fessenden, R. W. J. Am. Chem. SOC. 1960, 82, 766. (b) Cole, T.; Heller, C.; McConnell, H. M. Proc. Natl. Acad. Sci. U.S.A. 1959, 45, 525. (24) Derbyshire, W. Mol. Phys. 1962, 5, 225. (25) Venkataraman, B.; Fraenkel, G. K. J. Chem. Phys. 1956, 24, 737.

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