analysis of in vivo and in vitro esr spectra of 2h-flavin, an intrinsic spin label of flavoproteins

Volume 16, number 3 CHEMICAL PHYSICS LETTERS 15 October 1972

ANALYSIS OF IN VW0 AND IN VITRO ESR SPECTRA OF 2H-FLAVIN, .4N INTRINSIC SPIN LABEL OF FLAVOPROTEINS *

J-R. NORRIS and H.L. CRESPI Chemistry Division, Atgonrre Natimal Laboratory. Argonne, Illinois 60439, L&i

Received 25 July 1972

ESR experiments with fully deute rnted flavoptotcin have made possible the fast determination ofg_tensors and coupling constants for the radical form of this protein. Rotational diffusion correintion times for several in vitro and in viva fkwoprolcin systems have been calculated and the utility of the saturation behavior of *H-flatin radicals is demonstrated.

1. Introduction

Ffavoproteins containing ff avin mononucfeotide (FMN) and tfavin ade~ned~uc~eot~de (FAD) as prosthetic groups are among the most important electron transfer agents and oxidation-reduction enzymes in biochemistry [l--3] . The intermediate (“semiquinone”) oxidation state of these proteins may be in- vestigated by electron spin resonance [I ,4-61, and a detailed analysis of the ESR spctra of these proteins may provide significant insight into biologica! proc- e-s evolving electron transport, such as photosyn- thesis and respiration.

The ESR properties of special interest include the hyperfme tensor, the g-tensor, response to microwave power sa~rat~on, and the spin-label properties of the ESR-active flavin group. Although the Lotropic hyperfine coup!ing constants have been established for free FIMN and similar compounds [5,6], the hyperfine tensors for the protein-bound material have not yet been reported.

The ESR “powder spectrum” of ordinary ‘H- ff avoprotein is too compiicated and too featureless for reliabte determination of hyperfine tensors. A_ minimal analysis requires determination of two nitro-

* Work performed under the auspices of the tJ.S. Atomic En- ergy Commission.

gen and one hydrogen hyperfinc tensor and theg- tensor. The ESR spectra of flavoproteins in solution are even more difficult to analyze because of the effect of slow rotational diffusion on the anisotropic tensors. This spin-label property of the flavins has been severely limited in applications until now [5,7], because the hype&e culd g-tensors must be known in order to interpret qu~titat~ve~y such spectra These characteristics also make in vivo ESR observa- tions on fH-flavoproteins difficu!t.

We have prepared and characterized a fuUy deuterated flavoprotein whose “powder” ESR spectrum is relatively easify interpreted. 2H-ffavoprotein ESR spectra may be accurately simulated using only g- anisotropy and two nitrogen hyperfine interactions, m*&ing possible c’alculation of the effects of rotational diffusion on the spectra. The simple, but ,*lfficient- ly detailed ESR spectrum of 2H-flavoprotein, not only yields info~ation about “Jle binding of the flavin prosthetic group to the apoprotein, but provides a unique tool for the investigation of in vivo biological systems. We have, in fact, observed in vivo 2H-fiavo- protein via ESR while it functions in ar? electron transport reaction in the fully deuterated alga, Synechococ. cus lividus [8] , Rotational correlation times for the protein may be determined for 2H-fiavoproteins both in viva and in +ro, thus making 2H-FMN a very useful intrinsic spin label.

Volume 16, number 3 CHEMICAL PHYSICS LEITERS IS Oct+?r 1972

2. Experimental 3. Resuhs

The flavoprotein (mo!ecular weight, 17 OOG dalton; one FMN per molecule) was extracted from the ther- mophilic (SO’C) blue-green alga S. lividus [9]. This particular protein is a member of a class of fiavopro teins known as flavodoxins [3] which are known not to undergo reversible aggregation [ IO] . The neutral semiquinone free radical was formed anaerobically by photochemical reduction in neutral 2H20 phosphate buffer [ 11,121 . The ESR spectra are concentration independent, indicating that magnetic resonance es- change phenomena are not important.

3.1. Powder spectra Our ESR spectra! simulations employ the spin

hamiItonian:

~=~g-Hi~~ItNs’tNlo].l^, (1)

where only the g-tensor and two nitrogen hypcrfine tensors are included. By assuming the same axiai symmetry axis for the three tensors and fo!lowing Blinder [ 141, the position at which resonance occurs is defined by

Protein powder spectra were obtained by irnmo- bilizing the flavodoxin in a frozen buffer solution at - 160°C and by adsorption at room temperature to DE-52 (Whatman) ion-exchange cellulose (matrix protein). In vivo spectra were obtained using a thick slur- ry of fully deuterated algal cells. A Varian E line ESR spectrometer with an El 01 microwave bridge and a rectangular TE 102 cavity with quartz dewar insert was used in all ESR experiments. Liquid samples were contained under nitrogen in Scanlon S-808 aqueous sample cells. The modulation frequency was 10 KHz. g-values were determined by measuring the microwave frequency,with a 52451, Hewlett-Packard counter, and the magnetic field with strong pitch as a marker at g = 2.0028.

*, = h@“(g~cos*B +pZsin’B)] --l/2

+JI~+ {(A~, -BN5j2+3B (2.4 N5 Ns

+B x5

jc05*aP

+~‘~~lo{(~N,,-BN,0~2+3BN,o(WN,,+BN,o)cos2’)1’2,

where Bi s i(Ti-Ai). 0 is the angle between the ex- ternal applied magnetic field and the axial symmetry aXis. TN5 and TN,, are parallel hyperfine coupling constants for N, and NlO, respectively;ANs and ,4~,e are isotropic coupling constants;fifNN, and i%$r,e are nitrogen spin quantum numbers and can be used to iden- tify each of the nine lines defined by cq. (2);g,, and gl are the usuaI components of the g-tensor.

Spectra forg-value determinations and least square analysis were obtained by repetitive data accumula- tion in a Nicolet 1074 Fabritek (1000 data points), which was on line in a time-sharing mode to the ANL Chemistry Division Sigma 5 computer. The data were stored on disks and later all computer calculations were made in the Sigma 5 computer. The powder spectrum calculations employed a least squares analysis that automatically varied the intensity,g,, gl, AN,, ;(TN, -A&), +,,, ;(TN - ‘+ linewidth. Solution (rotational dipfusion

,-,I and the \ spectra

were simulated by “inverting” nine 30 X 30 complex matrices once for each field position of the spectrum. One thousand points were obtained over a range of 100 gauss using the time-saving method ofGordon and McGinnis [ 131.

In computer simulation of the powder spectrum, thirty equally separated angles of 0 were employed to locate each lorentzian spin packet of relative intensity sin8. The final agreement between calculated and experimental spectra, determined by least square analysis, is very good (fig. la). Tensors derived from this treatment are listed in table 1. Al! other hyperfine interactions are included in the lorentzian spin packet linewidths used in the calculations.

As would be expected, the center portion of the spectrum is the most troublesome to simulate accurately because of the omission of the nuclear Zeeman and quadmpole interactions. The wings of the spectrum are deterrr;n:d by paralle! components of the tensors and the center by the perpendicular compo nents. Determination of the isotropic coupling con- stant requires both T, and TL. Thus, g1 and the .4’s are perhaps not known as accurately asgn and the Ti’s.

At present we feel the uncertainty of the isotropic

(2)

543


$H2OPO;

(?HoH)3 732

the fact that :(7x1 +g,J obtained for the protein is identical to the isotropic value obtained for free FMN [ 151, even thoughgl is extracted from the center of our spectrum.

Low Power 3.2. Rotarioilal diffitsiorz effecrs

0 The precise simulation of the rigid medium spec-

Flovin hlononucleotide (FMN) trum clearly demonstrates rhat the basic difference between the free FMN ESR spectrum and the fully

7----- immobilized, low-power, protein spectrum is pro- duced by the anisotropic electron-nuclear dipole- dipole interaction and a small amount ofg-anisotropy. Both these factors average to = 0 in free FMN.

_ - F. ._.._ _ However, the slow rotational diffusion of flavodoxin B. Rlgid in solution will only partially average these anisotrop-

ic factors and spectral interpretation becomes more complicated. One method of quantifying the effect of rotational diffusion on the ESR spectrum of the protein is by measuring the field separation of the outermost peaks [I&-18]. We designate this quantity F.

The spin hamilionian employed above is quite accurate for the calculation of F. The two peaks that define F result from molecules with 0 = 0 and

C. Solution

-- Low Power

/w High Pobver

(Af--s,fiJ~,O) =(I, 1) or(-1,Ll). We have calcuIated this maximum splitting as a function of isotropic rotational diffusicn correlation time (fig. 2) Ising the method of Norris and Weissman [ 16]_ Re,:er,tly, McConnell and coworkers [ 171 have succtssfully applied this method to nitroxide spin labels. In spin- label appIications the method of Norris and Weissman [ 161 is much easier to apply than more rigorous methods [ 18-2 I] , yet essentially the same correlation

Fig. 1. (a) Solid Ike, the ESR “2,owder” sptxtrum of 2H-flavodoxin radical in frozen ‘Hz0 buffer. The microwave power was attenuated by 50 dB to minimize saturation; dotted line, simuhtion of this powder speckurn. (%I Fine line, high power (100 mW) spectrum of *H-flavodosin radical in frozen ‘Hz0 buffer; heavy line, high power (!OO mW) spectrum of the matrix-bound radical at room temcxature. (c) Spectra of 2H- flavodoxin radical in solution ai room tem_xrature at 0.05

mW (fine line) and at 1 C3 mW (heavy line).

coup!irg ccnstant is somewhat smaller inan the difier- ence between these constants for the protein and free FMN. Equally gocd simulated spectra are obtained when the A-values for the protein are varied from 0.2 G smaUer to 0.5 G larger ihan the valuei of table i. The overall validity of our treatment is supported by

time, ~c, is obtained [ 17,201 . In the slow motion limit (TV long), F is easily measured as the associated peaks are quite sharp. As 7c decreases, F decreases, and eventually the maxima in the extreme spectral wings are unresolved and F can no longer be measured. Computer calculations can define F for small 7c even though experimotitally obtained low power ESR spectra lack sufficient signal-to-noise to allow measure- ment of F. However, an experinental method for determination of Feven in unfavorable conditions can be established.

Flavoproteins are well-known for possessing un- usual microwave power saturation properties [7]. In general, the center of the spectrum saturates more easily than the wings, so that the parameter F is easily

15 October 1972 VoIume 16, number 3 CIiEhlICAL PHYSICS LETTERS

Table 1 ESR pyameters fcr ‘H-F&IN systems

System gll Sl =N, AN5 TNl0 “WI TZHs AZH 5

2Hz0 buffer a) 2.0032 2.0032 0 7.2 f 0.1 II 3.6 + 0.1 0 1.2 immobilized

fhvodosin b) 2.0018 2.0039 18.4 5.9 8.8 2.5 - -

a) The g-value is from ref. [ 151, for ‘H-FAIN in neutrsl phosphate buffer. A-vaiues xre from [ 121. The other (unlisted) hyperfinc interaction pnrrlmetcrs are too smaII to be measured [S] .

b) Only two hyperfine interaction tensors ilze obtiincd. Tlx values Listed are for frozen solution or frozen matrix. Vnlue for ma- tris protein at room temperature would be only = 0.5 percent smaller. As discussed in the test, uncertainties xc not giwn.

Fig. 2. The ckuhted separation F, of the extreme high field nnd low field peaks as a function of correlation time. The cll- culations ;LT~ based on isotropic rotational diffusion, a reason- nblc model since hydrodynamic mwsurements [ZO] show ti- vodosin to be essentially spherical. As ~c approaches = 5 nscc,

the c&uMions become invalid.

measured in saturated species. Because, empirically, F

increases slightly with increasing power, F can be extrapolated to zero power for comparison with the calculated values of F that do not include the effects of saturation. However, for typical in vivo systems, the low concentra:ion of flavoprotein may make extrap@ lation to zero power unreliable, and in this case one can compare Fs with saturated spectra of known solutions of flavoprotein.

Correlation times for flavodoxin in several systems have been obtained by these methods and are listed in table 2. For a rigid, hydrated (20%) sphere of 17000 dalton and partial specific volume 0.64 cm3/g [9] we

Table 2 Rotntional ccrrclation times for ‘H-flavodosin in various

‘I~20 systems

system 7c (nscc)

buffer 8.1 f 1 buffer (hydrodynnmic &culation) 7.3 iO% albumin 10.4 ?r 1 25% (v/v) glycerol 21 is S. Iividus cells 15-25 3) R rubrum cells 1 l-w)

a) Obtained from saturated sp~ctrs.

calculate [21] a rotational correlation time (in ‘Hz0 buffer) of 7.3 nsec. Hydrodynamic measurements on flavodoxins [ IO,23] indicate a low frictional ratio, so this protein is nearly spherical and the hydration fac- tor is unlikely to be greater than 20 percent. Our experimental value ofF for 2H20 buffer at room temperature fitted to the simulation p!ot (fig. 2) gives a correlation time of 8.1 f 1 nsec, in reasonably good agreement with the calculated value. The correlation times obtained for 10 percent albumin and 25 per-

cent (L/vj glycerol solutions of ilavodoxin radical (table 2) increase linearly with viscosity, as expected

[171.

4. Discussion

That our parameter F can, in fact, be obtained from saturated ESR spectra seems quite reasonable. The ESR peaks determining F are due to so-called “turning points” near 0 = 0 for the spectral fragments due to ( III) and (- 1, -1) ESR transitions. When rn& tional processes occur in a time shorter than I?“,, T,


of the saturation parameter, (yHf)Tt T2, is replaced by l/Au where Aw is a measure of the miving of spin packets by the motional process [7: 211. The motional scrambling of spin packets (such as a random walk in angle 0 about the surface of a sphere) is more like- ly to produce a change in 19 near the turning point 0 = 0 than near the turning point 0 = ~12. Thus, the group of spin packets near 8 = 0 have a smaller l/Aw than spin packets near 8 = rr/2 and are more difficult to saturate. This differential saturation effect will become manifest at high power in flavin radicals undergoing 2ny motional process. Since the tensors of table .I will produce seven lines (two of the nine lines disappear through degeneracy), we expect to see six turning points in spectra obtained at high microwave power (the seventh and centermost line has no hyperfme interaction in this treatment). In addition to ob- taining F values from the (1,l) aid (- 1,- 1) transition turning points, one should be able to analyze saturated spectra for derails of intramolecular motions of flavin radicals.

For esample, f!avodoxin adsorbed on cellulose DE-52 gives !ow-power room-temperature spectra al- most identical to low power spectra at - 160°C (fig. la). At high power in frozen solution (or frczen matrix) T, < r, sb that saturation destroys all spectral features (fig. lb). However, the ESR spectrum of room iemperature matrix flavodoxin at’high power shows six well-defined turning points because of mo- tio11 of the ESR-active part of the FMN prosthetic group. Since the matrix bound protein is undergoing essentially no rotational diffusion (its Fvalue is about one percent smaller than that of frozen matrix or frozen buffer), this spectral difference (fig. lb) is probably due to motion of FMN relative to the protein or to segmental motion of the prosthetic group. This datum also indicates that the T,‘S of table 2 are for the protein as the contribution to the ESR spectrum by local FMN motion is negligible for flavodoxin in solution. Fig. lc ihustrates the advantage of using saturated spectra to obtain an accurate measure of F: of radicals in solution.

5. Con&sion

Fully deuteratetl flavins constitute a new class of i&i&c spin label, as no chemical modification of

the flavin or flavoprotein is necessary to achieve the spin-label property. Thus, the active center of many flavcprotein enzymes can be studied under natural conditions by the quantitative ESR techniques de- scribed here. In principle, LH-flavin is a similarly useful spin label, but the ESR properties of the IH radicals make quantitative study very difficult. Under conditions of microwave saturation, “H-flavin radicals yield a wealth of spectra detail that can be related both to rotational diffusion correlation times and to intramolecular motions within flavoproteins. The radical form of the flavoprotein flavodoxin has been observed in live cells of the blue- green algae, S. lividus (a photoactive signal) and in the photosynthetic bacterium, Rhodospirillum rubmm (a non-photoactive signal). The parameter, F, as well as the general similarity ;lf the spectrum to spectra of in vitro flavodoxin solutions, indicate that flavodoxin functions in solution in’ both these organisms [ 241.

References

111

I21

131

[41 151

161

f’!

[81 PI

[lo1

Illi [I21

Ll3i

[I41 [W

1161

[I71

H. Knmin, ed., FIzzins and fLavoproteins (Univ. Park Press, Baltimore, 1971). A.H. Lehninger, Biochemistry (Worth, New York, 1970). A.H. Neims and L. Hellerman, Ann. Rev. Biochem. 39 (1970) 867. A.Ehre&rg, Arkiv Ke_mi I7 (1962) 97. F. Xfuller, P. Hemmerich, A. Ehrenberg, G. Palmer and

V. Massey, European J. Biochem. 14 (1970) 185. W.H. Walker, A. Ehrenberg and J.M. Lhoste, Biochim. Biophys. Acta 215 (1970) 166. J.S. Hyde, L.E.G. Eriksson and A. Ehrenberg, Biochim. Biophys. Act;\ 222 (1970) 688. J.R. Norris, H.L. Crespi and J.J. Katz, to be published. H.L. Crespi, U. Smith, L. Gajda, T. Tisue and R.A. Ammeraal, Biochim. Biophys. Acta 256 (1972) 61 i. I-!. Vetter Jr. and J. Knappe, 2. Physiol. Chcm. 352 (1971) 433. V. hfawy and G. Palmer, Biochemistry 5 (1966) 3181. H.L. Crespi, J.R. Norris and J.J. Katz, Biochim. Bio- phys. Acta 253 (1971) 509. R.G. Gordon and RP. hfd;irmis, J. Chem. whys 49 (1968) 2455. SM. Blinder, J. Chem. Phys. 33 (1960) 748. LE.G. Eriksson and A. Ehrenberg, Acta Chem. Stand. 18 (1964) 1437. ;R;9Xotis and S.I. Weisrnann, J. Phys Chem. 73 (1969)

RC McC;illey, E, J. S~himshick and .H. hf. McConnell, Chem. fiys Letters 13 (1972) 115.

Volume 16, number 3 CHEMICAL PHYSICS SETTERS I5 October 1972

[is] H. Siliescu and D. Kivelson, 3. Chrm. Phys. 48 (1968) LT. hiuus and P.W. Atkins (Plenum Press, New York, 3493. 1972) ch 13.

1191 S.A. Goidm=,G.V. Rruno, C-F. Polzaszekand J.N. Freed, J. Chem. Phys 56 (19’72) 716.

[29] R.G. Gordon and T. Messenger, in: Electron spin re- lax&ion in Liquids, eds LT. Muus and P.W. Atkins (Plenum Press_ New York, 1972).

[ 211 J.H. Freed, in: Electron spin relaxation in liquids, eds,

1221 C-P. Poole Jr. and H.A. Faracb, Rei;tuation in magnetic resonance (.4cademic Press, New York, 1971) p. 65.

123 J H. Bathe, P. Hemmerich and H. Sund, in: F&ins and ~avoprote~n~~ ed. H. &uni.n (Univ. Park Pies B&i- more, 1971) p_ 211.

j24] HI,. Crespi, J.R. Norris and J.J. K&z, to be published.

analysis of in vivo and in vitro esr spectra of 2h-flavin, an intrinsic spin label of flavoproteins

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