HARDEN M. MCCONNELL AND JAN C. A. BOEYENS
12
and interpreted quite thoroughly. Our experiments are conducted at fixed microwave frequency and variable magnetic field. There is a hope that transitions which are magnetically tunable and have some electric dipole intensity might be accessible. However, the electric dipole character in a magnetic field depends on the magnitude of the spin-rotation coupling which is rather small, and for this reason I am pessimistic about our chances of success. At the present stage we would prefer to concentrate on small molecules in triplet states, where the probability of success is somewhat higher. 1cI. C. R. SYMONS (University of Leicester, Leicester). Some time ago we published an esr spectrum of a radical formed by ultraviolet photolysis of C l 0 ~in rigid sulfuric acid, which we assigned to C1-0-0 formed by isomerization of 0-C1-0. We
ought to be able to make a clear choice by comparing your derived hyperfine coupling constants with ours and I wondered if you have yet obtained these from your data on C107 A. CARRINQTON. We have not yet completed the analysis of the C10 spectrum. With good fortune we should be able to obtain values of the hyperfine parameters describing the Fermi contact interaction and the dipolar interactions between the nuclear moment and the moments arising from spin and orbital angular momentum. The electric quadrupole coupling constant will also be obtained. It would then certainly be possible to calculate the appearance of the esr spectrum of C10 in a solid, given information about the strength and symmetry of the perturbation from the surroundings.
Spin-Label Determination of Enzyme Symmetry1
by Harden M. McConnell and Jan C. A. Boeyens Stauffer Laboratory for Physical Chemistry, Stanford, California (Received September $7, 1966)
The paramagnetic resonance of spin labels attached to protein molecules in single crystals can be used to detect noncrystallographic symmetry elements in these molecules. Such symmetry axes arise when proteins are built up from a number of symmetry-related equivalent subunits.
In previous work it has been shown that the paramagnetic resonance of synthetic free radicals (“spin labels”) can be used to probe the structure, motion, and chemical reactions of nucleic acids and proteins in solution.z-6 The purpose of the present report is to indicate briefly how one type of biologically significant structural information may be obtained from the paramagnetic resonance of suitably spin-labeled proteins in single crystals. Particularly useful spin-labeling molecules contain the protected nitroxide groupzb4
I
0
where the nitrogen atom is directly bonded to two tertiary carbon atoms. I n such nitroxide radicals the odd T h e Journal of Physical Chemistry
electron is localized almost entirely on the nitrogen atom. The groups R and/or R’ provide a means for attaching the radical to particular amino acid side chains of a protein, to active sites, to hydrophobic sites, etc. Because of anisotropy in the nitrogen nuclear hyperfine tensor? and the g-factor tensor,? the paramagnetic resonance spectra of the nitroxide (1) Sponsored by the Office of Naval Research under Contract NONR-225(88). (2) (a) S. Ohnishi and H. M. McConnell, J . Am. Chem. SOC., 87, 2293 (1965); (b) T. J. Stone, T. Buckmsn, P. L. Nordio, and H. M. McConnell, Proc. Natl. Acad. Sci. U . S., 54, 1010 (1965). (3) L. Stryer and 0. H. Griffith, ibid., 54, 1785 (1965). (4) 0. H. Griffith and H. M. McConnell, ibid., 55, 8 (1966). (5) L. J. Berliner and H. M.McConnell, ibid., 55, 708 (1966). (6) J. C. A. Boeyens and H. M, McConnell, ibid., 56, 22 (1966). (7) 0. H. Griffith, D. W. Cornell, and H. ;zI. McConnell, J . Chem. Phys., 43, 2909 (1965).
SPIN-LABEL DETERMINATION OF ENZYME SYMMETRY
spin labels are sensitive to the rate of molecular tumbling. Since typical enzymes in solution are large (e.g., mol wt lo4-lo5) and relatively rigid, their tumbling motion in solution can usually be neglected, and the molecular motion that affects the paramagnetic resonance spectra is the motion of the spin label relative to that of the enzyme.2b-6 Depending on the mode and site of spin-label attachment, the motion of the nitroxide group relative to the protein has been found to vary all the way from “strongly immobilized” to “weakly immobilized.” The resonance spectra in the former case are essentially identical with the “powder” spectra of nitroxide radicals in rigid glasses at low temperatures, whereas in the latter case the spectra are only slightly broadened relative to the sharp three-line spectra of nitroxide radicals in solution in the absence of protein (“mobile” labels). When proteins are crystallized, it may be safely anticipated that strongly immobilized spin labels will remain strongly immobilized, and weakly immobilized spin labels mill sometimes remain weakly immobilized, but sometimes weakly immobilized spins may become more strongly immobilized if crystal forces are large enough. We have recently shown that a nitroxide-maleimide spin label reacts with a high degree of specificity with the two symmetry-related SH groups on the 0 chains on horse oxyhemoglobin to give a strongly immobilized spin resonance signal in solution.6 I n subsequent work it has been found possible to observe the anisotropic resonance spectra of this immobilized spin label in single crystals of horse oxyhemoglobin.8 A molecular model of tht>labeled hemoglobin has been prepared, based on the paramagnetic resonance datas and the structure of hemoglobin deduced by Perutz and cow o r k e r ~ . ~Thi,s work strongly suggests that it will be possible to carry out similar studies on many other crystalline proteins, especially enzymes. The paramagnetic resonance spectra of spin-labeled protein crystals must necessarily show the symmetry of the crystal structure and the symmetry of the protein molecules on which the structure is based. If an element of crystal symmetry (e.g., a twofold rotation axis) coincides with a molecular symmetry axis, then the existence of this molecular axis can usually be determined rather simply using X-ray diffraction. Proteins in which twofold molecular rotation axes have been discovert?d in this way @-lactoglobulin,10lacticodehydrogenase 114,” and glyceraldehyde phosphate dehydrogenase.I2 If the protein molecular symmetry axis does not coincide with the crystallographic axis, then the discovery of this axis will in general be quite difficult, requiring much data collection and a significant par-
13
tion of a complete structure determination. Such axes should, however, be observable for spin-labeled crystalline proteins. For example, when two spin labels attached to a given protein are related by a twofold rotation axis, then the resonance spectra of the two labels will in general be different unless the applied magnetic field is parallel or perpendicular t o the axis. Clearly, the resonance method is capable of detecting such axes and is especially suited to those cases where the X-ray method is so difficult. We now consider the relevance of such axes to protein structure and molecular biology. Symmetry axes in proteins can be present when the proteins are built up from identical subunits (oligomers such as dimers, tetramers, etc.). The existence of such symmetry axes appears to be of great interest to theoretical molecular biologists. Thus, Crick and OrgelI3 have pointed out that the phenomenon of intracistronic complementation between mutants of the same protein suggest that the active (enzymatic) form of such proteins is an oligomer built up of subunits that are related to one another by axes of symmetry. ;\lonod, Wyman, and Changeux14 have presented even more general arguments and speculations for believing that oligomeric proteins have symmetry axes, especially the allosteric regulatory enzymes. I n fact, the majority of enzymes may be built up from subunits so as to have elements of symmetry. Since, of course, the individual amino acids are optically active, the symmetry operations of interest do not include inversions or mirror planes and will most likely involve one or more twofold rotation axes or perhaps rotation axes of higher symmetry. I n addition to the determination of symmetry axes, the paramagnetic resonance of suitably spin-labeled symmetrical enzymes can be used to obtain evidence of allosteric conformational changes due to the presence on the protein of substrates, inhibitors and/or activators, since such allosteric effectors should in general produce conformational changes that will modify the relative orientations of the principal axes of symmetry related spin labels. It is clear, therefore, that the paramagnetic resonance of spin-labeled single crystals of proteins offers the pos-
c.
(8) s. Ohnishi, J. A. Boeyens, and H. N.IlcConnell, Proc. Natl. Acad. Sci. u. 56, 809 (1966). (9) M. F. Perutr, & G. I. Rossmann, A. F. Cullis, H.Muirhead, G. Will, and A. C. T. North, A‘ature, 185, 416 (1960). (10) D.W. Green and R. Aschaffenburg, J. Mol. Biol., 1, 54 (1959). (1964). (11) B. Pickles, B. A. Jeffery, and M. G. Rossmann, ibid.. 9, 598
s.,
(12) H. C. Watson and L. J. Banasrak, Nature, 204, 918 (1964). (13) F. H.C.Crick and L. E. Orgel, J . Mol. Biol., 8, 161 (1964). (14) J. Monod, J. Wyman, and J. P. Changeux, ihid., 12, 88 (1965).
Volume 71, Number 1
January 1967
R. 0. C. NORMAN AND B. C.
14
sibility of testing some recent theoretical predictions in the field of molecular biology.
Acknowledgments. This research has benefited from facilities made available by the Advanced Research Projects Agency through the Center for Materials Research a t Stanford University.
GILBERT
protein structure. Minor distortions in the immediate vicinity of the label are of course inevitable. Work in progress by L. R. Berliner, J. C. A. Boeyens, and H. M. McConnell show that similar conclusions apply to a-chymotrypsin crystals spin labeled a t the active site.
Discussion
M. C . R. SYMONS(University of Leicester, Leicester). What is the distance between the nitroxide radical and the iron atom? Would one not expect to see some manifestation of the proximity of electron spins in some of the systems studied?
R. L. WALKER(Haverford College, Haverford, Pa.). Can the bulky label group interfere with normal folding of the protein chain to which it is attached? H. P*Z. ICZCCONNELL.The spin-labeled hemoglobin crystals were isomorphous with crystals of the native protein. From this we can conclude that there is no large-scale distortion of the
H. M. MCCONNELL.The distance between the radical and the heme iron is estimated to be in the 15-20-A range, and thus far we have not yet observed any effects to indicate an interaction between them. However, there is a reasonable chance that a spinspin interaction could be detected at low temperatures in methemoglobin.
Electron Spin Resonance Studies of Oxidation. IX.1
Some Interesting
Properties of Iminoxy Radicals
by R. 0.C. Norman and B. C. Gilbert Department of Chemistry, The University of Ymk,York, England
(Received September 67, 1966)
Analysis of the esr spectra of a wide range of iminoxy radicals, RR'C=NO., has enabled us t o elucidate the variations with structure of the hyperfine splitting constants of these u radicals. The results are in marked contrast with those for ?r radicals: in particular, coupling is maximal for substituents in the molecular plane, long-range transmission of spin density occurs through the u bonds, and in certain radicals with the appropriate geometry a direct orbital overlap is postulated. The direct overlap seems particularly effective for fluorine? chlorine, and bromine, for which remarkably large splittings are observed. Studies of the radicals from ortho-substituted benzaldoximes and acetophenone oximes, including the temperature and solvent dependencies of aF in the anti-iminoxy radical from o-fluoroacetophenone oxime, lead to conclusions about the conformational preferences of these radicals.
Introduction There have now been a, number of electron spin resonance studies of the iminoxy radicals, RR'C=NO., formed by the abstraction of a hydrogen atom from oximes. The radicals have been generated from the oximes by irradiation in the solid state2p3and by oxidation in solution using ceric ion4or lead tetraa~etate.~-' The J O U Tof~Physical Chemistry
Iminoxy radicals have been characterized as u radicals. The unpaired electron is contained in an or(1) Part VIII: A. L. Buley, R. 0. C. Norman, and R. J. Pritchett, J. Chem. SOC.,Sect. B , 849 (1966). (2) I. Miyagawa and W. Gordy, J. Chem. Phys., 30, 1590 (1959). (3) y. Kurita, M. Kashiwagi, and H. Saisho, 86, 578 (1965).
J. Chem, sot, Japan,
