Bhinyo Panijpan Department of Biochemistry Mahidol University Rama VI Road Bangkok 4, Thailand
Chirality of the Disulfide Bond in Biomolecules
To the chemist, the sulfur-sulfur hond is a functional group ofdiverse rt,artivity and ~nterestingstere~,chemisrry. l'u thr hiochemisr. the 5-5 is an easily clen\,ed linkage which is an intliipens;thle structural feature 01' most proteins and polypeptides and is present in sume smalll!r i)iomolccules. Ohviously, n concerted effort by the c h t m ~ sand t the biochemist will mure rapidly bring ahuur a hettrr understanding of the physiwl p r o p t ~ t i t . ~chemical , hehavior, and hioluaicd roles of thedisulfidc bond Thisarticleisintended tudrnw theartmtion o i hoth rrouDs utscientists to the chirillit\ oi the disulfide hond a n i its ;ignificance in the structure and activity of hiomolecules. It was predicted, as long ago as 1934, thatcompounds conwith untaining groups of the general structure, -A-Bshared electrons on the two adjacent atoms, should have skewed configurations (1). The -S-Sgroup is one such group. Structural studies, mainly by X-ray diffraction and chiroptical techniques, have established the chiral spatial arrangement of the disulfide bond, e.g., in H2S2, in allotropic forms of the elemental sulfur and in various organic comwunds. Recentlv. a meat deal of interest has been eiven to the " Lhirality of the disulfide linkage in molecules oT biological importance (2-5). These include lipoic acid, a coenzyme; cystine, an amino acid (see Fig. 1);aderivative of the antibiotic eramicidin S (6): . elutathione disulfide. an oxidized form of a ubiquitous tripeptide thiol(7); malformin A, a mycotoxin; oxvtocin and vasopressin, both small . polvpeptide hormones, .. . and several globular proteins, e.g., lysozyme, ribonuclease, rhymotrvpiin. and carhoxvpeptidase. Apart trum t h w , there are other lwsrr-known classes of disulfide ;rind s~~IFnylryl compounds which 1 wid serve ro elucidate turther the rule and reactivity of the S-S grnup in hiolugical sysrenls t&-121. The C-S-S-Cmuirly occurs in hi~mdeculeshaving thr disultidc linknur. Tht, 5-Stwnd leneth is tvoirallv .. .between 2.114 and 2.06 A, while the
COOH COOH i i H2N-C,,-H v-C,,-H
I
i
CH2-s-S-CH2
/C"Z.~
Hzc
I
\ & / IS (cH~)~
I
L-cystine
COOH
(1a )
6.8-Lipoic acid (I b)
Figure 1. L-cystine (a)is an amino acid formed by two L-cysteinyl residues. The two asymmetric a-carbons have identical configuratian.6.8-Lipoic acid ( b )is acoenzyme. The ring structure is a 1,2dithiaianeringhavinganeasymmetric carbon (C').
.,
v
hond angle usually ranges between 100° to 108'. The disulfide dihedral anele ( a ) is the anale subtended by the two C-S bonds when'thes-s group isviewed end-on from either direction (see Fig. 2). The screw sense, as distinct from the magnitude, is also important, e.g., +a and -a are of equal magnitude hut opposite screw sense. If only one preferred dihedral angle is maintained as in 2(a), the molecule is chiral, since it cannot he superimposed upon its mirror image (2(b)) and vice versa. If a collection of one c o m ~ o u n dcontains an excess of molecular species with one preferred set of dihedral angles over other species with a set of dihedral angles of the opposite sense, then it would exhibit optical activity. This ootical activitv is inherent to the C-S-S-C linkace ((6.13) and c k be considered separate from other chiral fea&res that may exist elsewhere in the molecule; e.g., the optical activity of the chiral disulfide in L-cystine is distinct from that arising from the configuration of the a-carbons. Barring large hindrances to rotation around the S-S, the preferred dihedral angle is about 90'. Bonding constraints and steric factors, e.g., ring size, may cause this angle tovary from 90' and cause a change in S-S hond length and strength as a consequence. A t a of 90°, the repulsion between the lone 2p,-electron pairs of the sulfur atoms is a t a minimum. An increase in the 670 / Journal of Chemical Education
Figure 2.
The CS-SC dihedral angle has screw sense,righthnded or positive
(+a)as in (Zbl and ten-handed or negative (-a) as in (2a). These two skewed conformation^ are mirror
images and do not superimpose upon each other.
degree of overlap of unshared pairs of 2p-electrons on one st~lfurwith the 3d orbitals of the other sulfur would lead to an increase in the partial double hond character of the S-S linkage, i.e., shorter bond length and greater hond strength (14-17). The energy harrier to the rotation about the S-S linkaee from @ o f90' to 0" has been found to be 3-14 kcall mole2epending on the suhstituents on the two sulfur atoms (17.18). In carbocyclic compounds, the disulfide hond with a of about 90° can only be maintained in large rings. With decreasing ring size, say, from 7 to 6 to 5, becomes smaller and the strain increases (19) For the five-membered dithiolane ring in lipoic acid is about 21°. With such a dihedral angle, the p,-d, conlribution to the partial double bond of the S-S disappears and near maximum repulsion of the 2p,electroni bccurs. Incidentally, it is n o t known for certain whether the bond formed between the two "normal" divalent sulfur atoms is due to an overlap between two pure p or sphybridized orbitals. This is because the hond length gives no indication of the nature of the hond. The disulfide linkage has a characteristic absorption band between 250 and 380 om. The molar extinction coefficient (E) a t the wavelength of maximum absorption (A,.,) is between 100 and 500 and, thus, is comparable to phenylalanine E27anrn and about ten times lower than tryptophan E280nm(71). Eh,, and A, in this range are sensitive to the dihedral angle a and the S-S bond length (20). In the thioctic acid model studies, .\, is h g e r and El , is hwer ifis (I, d~xrrnsrs;r . ~ in, the five-memherea ring d l ; , 8-thiuc~ic acid (liprjic acid!, A,,,,, = n25 nm and E.,.,+... ... ....... = ]Xu, whereas in the seven-memhered ring, 4,8-thioctic acid, A,, = 250 nm and E250nrn = 490. The structure-de~endentuv ahsor~tionand the chiralitv of the C-S-S-C moiety render this group amenable to stidies by uv-spectrophotometry, circular dichroism (CD), and the re~~~~~
~
~~~
~
~
~~
lated optical rotatory dispersion (ORD). Simple S-S containing organic compounds and hiomolecules have been used to establish the relationship between the C-S-S-C hond parameters and the signs and amplitudes of the Cotton effects in CD and ORD. In 1,2-dithiane ring systems, it has been is asconcluded that a positive CD hand a t the longest A, sociated with a positive and a negative CD hand is associated with a negative (21). The spacing of the CD bands of chiral disulfides is largest when = 0 (22). Since hond strength and hond length also reflect the magnitude of +, vibrational spectroscopic techniques, e.g., laser Raman spectroscopy, should provide information on the +, especially, in cases where the uv absorption and chiroptical techniques cannot clearly do so (2,23-27). This situation usually arises when proteins are studied because of the high background absorption and optical activity due to the aromatic side chains of amino acid residues mentioned above. It is the S-S of the cystine residues that plays an important role in maintaining the specific molecular conformations of most S-S containing proteins. Cleavage of most or all of the disulfide honds usually leads to the disappearance of the native conformation and hioloeical activitv. and its de.. Cvstine . rivalives i n solid nnd solutim~have htwn investignt~dhy X-ray diffracrim ilnd chirtmtical techniaues. 'l'he ~Jisull'ldesrrrw sense in hexagonal L-cystine crystals is positive or righthanded (28). that in L-cvstine hvdrochloride. elvcvl-L-cvstine ant1 L-ryatine hvdrohrokitlt, is lert-hnndrd.ln soiuiion: both I.-cvatine 17. 311) and I.-holnorvstint: t,xl~ihitCottun eiierrs (see (29) for references). ~ x c e G for cystine in acid, the negative maxima occur a t wavelengths longer than the characteristic 243-248 nm absorption maximum shown by aliphatic disulfides and by cystine itself (whose absorption curve exhibits a shoulder) (Fig. 3). Near neutrality both the CD move to longer wavelengths. The character maxima and A,, of this cystine optical activity band is still the subject for argument. The high optical rotation of the disulfide in Lcystine, which has heen taken as evidence for a chiral disulfide grouping by some authors, is thought to be due toaccumulated optical rotatory power from three staggered rotamers in solution by others (31). But an inherently dissymmetric disulfide chromophore explanation is adopted for interpretation of the near-uv CD of disulfide-bridged peptides and the temperature dependence of the CD of cystine derivatives is accounted for by changes in the dihedral angle (30). The [2glycine] oxytocin (32) and trypsin inhibitor of adzuki beans (33). where there is no aromatic residue a t all, the positive hand about 250 nm must unequivocally he associated with a disulfide transition. The CD spectrum of bovine neurophysin
+
+
+
I1 is also of interest in this respect (5).T h e position of the positive hand strongly indicates that the dihedral angle is close to 90'. The crystal structures of lysozyme, ribonuclease, chymotrypsin, and carhoxypeptidase have been established and all the disulfide bonding parameters are known. In these proteins packing requirement may result in dihedral angles that vary substantially from the widely accepted value of 90' for L-cystine in solution. In chymotrypsin, e.g., this angle of the different disulfides ranees from 87-126' (34) and in Ivsozyme two disulfides h a v e = +100°, and otheitwo -1000 (35). In solution, ellipticity bands of disulfide-containing proteins occur a t wavelengths longer than 240 nm and the origin of part of the optical activity here is due to the CS-SC chirality (may he CC-SS chirality also). Attempts have been made to assien + t o individual disulfides in some of the nroteins studiel by CD with some degree of success. As mentioned earlier, the aromatic side chains usually contribute sizeahly to the absorption and optical activity spectra arising from the -CSSC- in the 250-330 nm region, making such studies less fruitful. Raman spectroscopy of solids and solutions of proteins has been used to partially overcome this problem, since S-S and C-S stretching bands occur in a region, e.g., 450-800 cm-', that is relatively free from other intense bands. In an earlier investigation on several simple organicand biochemical compounds with similar CC-SS dihedral angles, in solid phase, it was found that the S-S stretching frequency, u(S-S), varied linearly with the CS-SC dihedral angle previouslv estimated bv either X-rav or neutron diffraction or uvahiorption (3). ~ c linear e dependence of the S-S stretching freauencv on dihedral anele led to an estimate of a dihedral angle t i r millformin CS.SC, that was in t.xcellent agreement with that 65" (36). Recently a reinvestigation of the dependence of the values of S-S stretching frequency on the conformation about the C-S and S-S bonds of a wide ranae of disulfides helped solve this controversy and others and bring about a better understanding of the vibrational properties of the CCSSCC moiety (37-39). A new relationship between v(S-S) and has thus been proposed to take account of the new findings. Now a linear relationship can be conceived between "(S-S) and +from &60° (39). At values higher than 60°, the curve flattens rapidly (Fig. 4). In the light of these findings,
+
+
+
+
480
211
230
251
WAVELENGTH
270
211
/ 0
40
80
i
(nm)
Figure 3. The uv absorption spectrum of L-cystine in acid solution (dotted line) and the CD soectrum lsoiid line). The molar extinction coefficient scaie is !haarimmic. whereas the m i a r eilioticitv scaie is linear. Note the neaatke CD band at wavelengms longer than 240 nm and the absormm shoulder around 245 nm on the uv spectrum See (301 and ( 7 1 )
CS-SC dihedral angle Figue 4. Relationship between me Raman S-S stetching frequency of primary disulfides and the CS-SC dihedral angle. n(S-S) varies linearly with +between 0" and 60' and becomes quite insensitive to 60' $ < < S o . (Adapted from
