J . Phys. Chem. 1989, 93, 6381-6387
638 1
at the surface increased. In addition, they31 found that the rate oxidation of Fe(II), by SO5'- or SO?- and S(IV) by 02, and (iii) of electron transfer from the reduced methylviologen radical is the Fe( III),,-catalyzed autoxidation of S(IV).57 governed by the free energy difference between the redox potential Leland and BardI6 found that the rate of photooxidation of of the redox couple in the electrolyte and the Fermi level in the sulfite in the presence of iron oxide polymorphs varied by 2 orders solid and by the electrostatic interaction between the reduced of magnitude with the relative order of y-FeOOH > a-Fe2O3 > radical and the electrical double layer. The electrostatic effects y-Fe203 > 6-FeOOH > P-FeOOH > a-FeOOH. They attributed between the charged electron donor and the particle surface were this relative order in the S(IV) oxidation rate to differences in found to be the dominant factor contributing to the observed rate the crystal and surface structure as opposed to differences in constant. Somewhat similar results had been noted previously reactive surface area, hydrodynamic surface diameter, or band by Stramel and Thomas.78 gap. In oxygenated system they found no Fe2+released to solution after photolysis of S(IV) at pH 4.1. Meisel and c o - w ~ r k e r s ~ ' ~ ~ ~ Acknowledgment. We gratefully acknowledge the financial have recently studied the dynamics of charge transfer from reduced support of the US. Environmental Protection Agency methylviologen radicals to Fe( 111) oxides and oxyhydroxide colloids (R8116112-01-0 and R8113326-01-0). We are also grateful for in aqueous suspensions. They found that a fraction of the electrons the assistance provided by Drs. Liyuan Liang and Claudius transferred to the a-Fe203colloids were able to migrate into the Kormann. particle interior and form stable Fe304. The remaining fraction of transferred electrons formed Fe(I1) which was either released Registry No. Fe20,, 1309-37-1; S032-,14265-45-3; SO:-, 1480879-8; SZOt-, 14781-81-8; 0 2 , 7782-44-7; Pt4+,22541-31-7. to the bulk solution or adsorbed to the particle surface; as the particle size and pH decreased, the fraction of electrons trapped (78) Stramel, R. D.; Thomas, J. K. J . Colloid Interface Sci. 1986, 110, 121-129.
(79) Peri, J. B. J . Phys. Chem. 1965, 69, 220-230. (80) Kawakami, H.; Yoshida, S. J . Chem. SOC.,Faraday Trans. 2 1985, 81, 1117-1127.
Formation and Stability of Intramolecular Three-Electron S:.N, S:.S, and S:.O One-Electron-Oxidized Simple Methionine Peptides. Pulse Radiolysis Study
Bonds in
K. Bobrowski* Department of Biophysics, Institute of Biochemistry and Biophysics, Polish Academy of Sciences, Rakowiecka 36, 02-532 Warsaw, Poland
and J. Holcman* Accelerator Department, R i m National Laboratory, DK 4000 Roskilde, Denmark (Received: November 16, 1988)
Intramolecular sulfur-sulfur (S:.S)+ and sulfur-nitrogen (S;.N)+ three-electron-bonded radical cations and sulfur-oxygen (S:.O) radicals have been generated in aqueous solutions of some simple di-, tri-, and tetrapeptides containing methionine units due to oxidation by hydroxyl radicals under pulse radiolysis conditions. All these transient species are formed at the diffusion-controlled rate (k I 10" dm3 mol-' s-I), and they exhibit optical absorptions with the maxima at 390 nm (S:.Nand St.0-bonded species) and at 490 nm (S:.S-bonded species) with extinction coefficients of 5000-7000 dm3 mol-I cm-I. In slightly acidic solutions of tri- and tetrapeptides, a protolytic equilibrium between S:.O- and S;.S-bonded species was observed. The position of this equilibrium shifts by approximately 2 pK units when going from L-Met-Gly-L-Met (pK = 3.05) to L-Met-Gly-L-Met-L-Met(pK = 5.15). Conversion of the S:.O-bonded species into the S:.S-bonded species proceeds via kinetically distinct [H+]-dependent ( k = 107-108 dm3 mol-' s-I) and [H+]-independent ( k lo4 s-I) routes. In the pH range 6.0-9.0, a pH- and buffer-concentration-independent conversion of the 490-nm into the 390-nm absorption band was observed. This fast process ( k > lo's-') is consistent with the conversion of the S:.S-bonded species into the S;.N-bonded species.
Introduction In the past few years, a growing interest in oxidation processes in biological systems has stimulated several investigations of the radiolysis of sulfur-containing amino acids.l-4 It has been shown5 i n the dimeric mixed-valence complex [(R~(NH,!,),( 1.5DTC0)]5+that two neighboring thioether groups facilitate electron transfer in a way that is similar to that in compounds (1) Packer, E. J . J . Chem. SOC.,Perkin Trans. 2 1984, 1015. (2) Buchanan, J. D.; Armstrong, D. A. Int. J . Rodiat. Biol. 1976,30, 115. (3) ill^^, K,-o,; Masloch, B,; Gab], M,; A ~K,-D,~J , A ~~Chem, , ~ sot,, 1981, 103, 2734. (4) Davies, M. J.; Gilbert, B. C.; Norman, R. 0. C. J . Chem. Soc.. Perkin Trans. 2 1983, 73 1. ( 5 ) Stein, C. A.; Taube, H. J . Am. Chem. Soc. 1978, 100, 1635.
where the thioether group is adjacent to another lone-pair donor such as carboxyl or hydroxyl.6 Moreover, interaction of the oxidized sulfur center with the latter groups can result in a lowering Of the reduction potential which, in the case of 2,6-endo,endoand 2,6-exo,endo-thiomethylcarboxylate derivatives of norbornane, et a1.6 was confirmed Similar effects are expected to occur in proteins. They might well evolve from more efficient charge stabilization due to conformational changes leading to favorable mutual positioning of the electron donor-acceptor pairs' A change resulting in a substantial decrease in the reduction potential of (6) Glass, R. S.; Duchek, J. R.; Klug, J. T.; Wilson, G. S. J . Am. Chem. SOC.1977, 99, 7439.
0022-3654/89/2093-6381.$0l.50/00 1989 American Chemical Society
6382 The Journal of Physical Chemistry, Vol. 93, No. 17, I989 the radical intermediate has been invoked to elucidate the catalytic mechanism of yeast cytochrome c The relaxed radical-site conformation with lower reduction potential was characterized as the nucleophilic-stabilized methionyl thioether radical, with either the second methionyl sulfur or the carboxylic oxygen (aspartate residue) more closely positioned, while the mutually displaced nucleophilic centers characterized the conformation of the resting-state enzyme. A substantial amount of data on the stabilization of oxidized sulfur centers has been reported. Musker et al.loJ1were able to stabilize radical cations and dications formed during oxidation of mesocyclic and acyclic dithioethers in aprotic solvents; they were then able to evaluate some of their chemical properties. Asmus and c o - ~ o r k e r s ~studied , ~ ~ - ~the ~ oxidation of thioethers, thioamines, thio acids, and methionine by hydroxyl radicals generated by pulse radiolysis in aqueous solution. They showed that one-electron oxidation of sulfur atom led to the formation of three-electron bonds by coordination with a second sulfur or with other nucleophilic heteroatoms such as 0, N, Cl, Br, and I. The ease of formation and stability of a three-electron-bonded species depends on the conformation of the system, which affects the extent of orbital overlap, and also on the electron-donating withdrawing ability of both the substituents on S and the counterpart atom (X). Generally, in S:.X-bonded species the stability of the three-electron bond decreases with increasing differences between the electronegativity of S and X . The relative stability of S:.N, S:.S, and S:.O bonds is of particular concern in the oxidation of biomolecules, e.g., peptides, redox proteins, and enzymes, containing besides sulfide also amino and carboxyl functions. In our previous paper1* concerning methionine dipeptides, we have reported that the formation of S:.N bond is effectively inhibited where S and N atoms are separated by the peptide bond. Recent N M R studies have shown that methionine is one of the most flexible protein amino acids.19 Since intramolecular interactions involving the methionine sulfur may be of importance for electron-transfer reactions and conformational fluctuations in the proteins, it is of interest to study the transient spectral and acid-base properties of one-electronoxidized species derived from methionine peptides. The present paper is a continuation of our interest in generation and stabilization of oxidized sulfur centers in methionine peptides. We report results obtained from pulse radiolysis of some simple methionine peptides containing varying numbers of methionyl, glycyl, and alanyl units.
Experimental Section The pulse radiolysis experiments were performed at Rim's 10-MeV H R C linear accelerator. The dose was determined with the ferrocyanide dosimeter using qZ0= 1000 dm3 mol-I cm-' and G = 6.0. The applied dose was 10-30 Gy in a 1-ps pulse. The optical detection system consisted of a 150-W Varian high-pressure xenon lamp with increased intensity in short pulses, a Perkin-Elmer double quartz prism monochromator, and Hamamatsu R955 photomultiplier. The data were recorded on a Nicolet Explorer (7) Hoffman, B. M.; Roberts, J. E.; Brown, T.G.; Kang, C. H.; Margoliash, E. Proc. Nail. Acad. Sci. U.S.A. 1979, 76, 6132. (8) Hoffman, B. M.; Roberts, J. E.; Kang, C. H.; Margoliash, E. J . Biol. Cfiem. 1981, 256, 6556. (9) Ho, P. S.; Hoffman, B. M.; Kang, C. H.; Margoliash, E. J . Biol. Cfiem. 1983, 258, 4356. (IO) Musker, W. K.; Wolford, T. L.; Roush, P. B. J . Am. Cfiem.Soc. 1978, 100, 6416. (1 1) Musker, W. K.; Wolford, T. L. J . Am. Cfiem.SOC.1976, 98, 3055. (12) Asmus, K.-D.; Bahnemann, D.; Fischer, Ch.-H.; Veltwisch, D. J . Am. Cfiem. SOC.1979, 101. 5322. (13) Hiller, K.70.; Asmus, K.-D. J . Phys. Cfiem. 1983,87, 3682. (14) Asmus, K.-D.; Gobl, M.; Hiller, K.-0.; Mahling, S.; Monig, J. J . Chem. Soc., Perkin Trans. 2 1985, 641. (15) Monig, J.; Gobl, M.; Asmus, K.-D. J . Cfiem. Soc., Perkin Trans. 2 1985, 647. (16) BonifaCiC, M.; A m u s , K.-D. J . Org. Cfiem. 1986, 51, 1216. (17) Gobl, M.; BonifaEiE, M.; Asmus, K.-D. J . Am. Chem. Soc. 1984, 106, 5984. (18) Bobrowski, K.; Holcman, J. fnt. J . Radiat. B i d . 1987, 52, 139. (19) Keniry, M. A.; Rothgel, T. M.; Smith, R. L.; Gutowsky, H . S.; Oldfield, G.Biochemistry 1983, 22, 1917.
Bobrowski and Holcman
4
I 0021
I
0011
1
11
, 300
LO0 L-nm
500
Figure 1. Optical absorption spectra recorded 4 N20-saturated aqueous solutions of lo4 mol
600 ps after the pulse in
L-methionyl-Lmethionine a t (a) p H 1, (b) p H 5.2, (c) p H 2.9, and (d) p H 2.9 (but 7 4 ps after the pulse). Inset: Yield of optical absorption (normalized to the actual OH radical concentration) recorded immediately after the pulse a t 490 ( O ) , and 390 nm (A), a s a function of p H in pulse-irradiated N,O-saturated aqueous solutions of lo4 mol dm-.' L-Met-L-Met; dashed curve, theoretical yield of the optical absorption a t 490 nm resulting from the complementary replacement of the S:.N-species by the S:.S-species. Dose 13 Gy.
111 digital oscilloscope, stored on disks, and processed on an on-line PDP-8 computer.20 Some pulse radiolysis experiments were carried out at the Notre Dame 7-MeV A R C 0 LP-7 linear accelerator in a flow cell (quartz, 1-cm path length) using 10-ns electron pulses with the dose 5 Gy/pulse. The description of the computer-controlled pulse radiolysis setup and data collection system is available else-
Extinction coefficients (eqgo) of the intramolecularly S:.Sbonded radical cations derived from L-Met-L-Met-L-Met, LMet-Gly-L-Met, and L-Met-Gly-L-Met-L-Met were determined from the relative absorbances of ClT and S:.S-bonded species in aerated solutions with or without peptides. Since for the all Met-Met dipeptide isomers reaction with Clz- can be expected to lead to the partial formation of S:.Cl-bonded specie^,^^*^^ therefore extinction coefficients were calculated from the intramolecularly S:.S-bonded radical cations absorption, assuming the yield of the latter to be equal to 80% of the total available OH radicals that were found for m e t h i ~ n i n e . ~ All solutions were prepared with triply distilled water, Lmethionyl-L-methionine and L-methionyl-L-methionyl-Lmethionine from Serva, L-methionyl-glycyl-L-methionineand L-methionylglycyl-L-methionyl-L-methionine from Research Plus Inc. and Bachem Feinchemikalen AG, L-methionyl-L-alanyl-Lmethionine from Research Plus Inc., D-methionyl-L-methionine, L-methionyl-D-methionine, D-methionyl-D-methionine, and Lmethionyl-L-methionyl-L-alanine from Bachem Feinchemikalen AG, and all were used as received. All other chemicals, HCI04, NaH2P04, and Na2HP0,, were of the highest commercially available purity. (20) Sehested, K.; Holcman, J.; Hart, E. J. J . Pfiys. Chem. 1983.87, 1951. (21) Patterson, L. K., Lilie, J. f n t . J . Radiat. Phys. Chem. 1974, 6, 129. (22) Schuler, R. H.; Buzzard, G. K. f n t . J . Radiat. Phys. Chem. 1976,8, 563. (23) Schuler, R. H . Document No. NDRL-2687 from the Notre Dame Radiation Laboratory. (24) BonifaEiE, M.; Asmus, K.-D. J . Cfiem.SOC.,Perkin Trans. 2 1980, 7 -I. 58
(25) Hiller, K.-0.; Asmus, K.-D. fnl. J . Radiat. Biol. 1981, 40, 583.
Intramolecular Three-Electron Bonds in Met Peptides The solutions (pH 0-3) were deoxygenated by purging with high-purity argon or nitrogen, and the solutions (pH 3-9) were saturated with high-purity NzO in order to convert enq-into OH, &e,< NzO) = 9.1 X lo9 dm3 mo1-I Hydrogen atoms are known to react with methionine with a rate constant at about lo9 dm3 mol-' S-I.~' The products, however, should not interfere with the optical signals observed in the range of interest, since earlier measurements showed only weak absorption below 300 nm in acidic solutions of m e t h i ~ n i n eand ~ ~N-acetylmethionine.28 ~~ These assumptions were confirmed by comparison of the spectra obtained in oxygenated (where essentially all the H radicals were scavenged by 0,) and deoxygenated solutions. All experiments were carried out at room temperature.
The Journal of Physical Chemistry, Vol. 93, No. 17, 1989 6383 (W-M)
+
Results and Discussion A . L-Methionyl-L-methionine.The reaction of OH radicals with L-Met-L-Met has been investigated in pulse-irradiated Ar, N2, or N 2 0 saturated aqueous solutions containing lo4 mol dm-3 L-Met-L-Met. Depending on pH, different transient optical absorption spectra were observed. The spectrum at pH 1 (curve a, Figure l ) , recorded 4 ps after the pulse, exhibits an intense band = 490 nm, and a smaller band in the UV, ,A, in the visible, ,A, = 290 nm. The 490-nm peak shows characteristics typical for three-electron-bonded sulfur-centered radical cations, e.g., structureless and very broad absorption band with a half-width of 1 eV and a maximum in the range 460-530 nm1.3929Assuming the yield of the intramolecular S:.S-bonded sulfur radical cation at this pH to equal the yield of the primarily formed S:.OH adduct (reaction I), which is expected to amount to -80% of
-
H,~;cH-CO-NHCHCOOH I I y 2 y 2 YH2
YH2
S
S I CH3
I CH,
'OHla'
H -&-
'
(W
(W The 290-nm band is attributed to the a-(alky1thio)alkyl radicals (11) by analogy with species observed in the oxidation of aliphatic s~lfides.~~.~~ The bimolecular rate constant for reaction 1, kl = (1.2 f 0.2) X 1Olodm3 mol-I s-l, was measured from the pseudo-first-order growth of optical density at 490 nm for L-Met-L-Met concentrations 2.2 X 10-5-2.0 X lo4 mol dm-3 (Table I). At pH 5.05 the transient spectrum (curve b, Figure l), recorded 4 ps after the pulse, shows a strong band with maximum at 390 nm and a weaker one at the low-wavelength side (290 nm). The latter is attributed again to the a-(alky1thio)alkyl radicals (11). It has been found that the OH radical induced oxidation of 3-(methylthio)propylamine, 3-MTPA, leads to the transient intramolecularly S:.N three-electron-bonded radical cation which = 385 nm.14 is characterized by optical absorption with A, Considering shape, width, and position of the absorption maximum and the possibility of an oxidized sulfur atom to be stabilized by a p-electron pair from nitrogen of the amino group, we assign the 390-nm band to the S:.N-bonded radical cations. In analogy with a recently outlined mechani~m'~ of the oxidation of 3-MTPA, the S.-.N-bonded radical cation can be formed via dehydroxylation of the primary O H adduct to a sulfur atom assisted by an intramolecular proton transfer from the NH3+ - group (reaction 3).
H3;n'-H -I S
S
I
I
.*.
OH
I
CH,
t
mm-
I 3p.gI
H 3 N ~ I T C O O -
(Met-Met)
1
( Met - Met ) 'OH
la 'OH
(3)
"."nCWH H3NncmH +
+
OH.'.S
H , ~ ~ C O O H
H , ~ ; ~ , C O O H
S
I
S
I
I
I
(111)
OH
.*.SI
s
S
s+
I
I
1
I
lb + H + , - H 2 0 lC'
H , & ~ , C O O H
JI
H & ~ , C O O H
-
L
st I
s I
s..s I
I
(1)
G ( 0 H ) = 2.730 that was found for m e t h i ~ n i n e ,G(S:.S)+ ~ of -2.15 can be estimated. The remaining 20% is expected3 to react via the hydrogen atom abstraction to yield a-(alky1thio)alkyl radicals (reaction 2). The Gt = 1.19 X lo4 dm3 mol-' cm-' calculated from the optical absorption at 490 nm in spectrum a in Figure 1 yields the extinction coefficient of (I), t = 5500 dm3 mol-' cm-I, which compares will with a value of t of 5000-7000 dm3 mol-' cm-' for (RzS:.SR2)+ radical cations.29 Janata, E.; Schuler, R. H. J . Phys. Chem. 1982,86, 2078. Mee, L. K.; Adelstein, S.J. Radiat. Res. 1974, 60,422. Lichtin, N. N.; Schaffermann, A. Radiat. Res. 1974, 60, 432. Asmus, K.-D. Arc. Chem. Res. 1979, 12, 436. (30) Farhataziz; Ross, A. B. Selected Specific Rates of Reactions of Transientsfrom Water in Aqueous Solution. Part III. Hydroxy Radical and Perhydroxyl Radical and Their Radical Ions; National Bureau of Standards: Washington, DC, 1977. (26) (27) (28) (29)
It is reasonable to assume that both sulfur atoms are attacked by the OH radicals with the same efficiency and that the 1:4 ratio of the hydrogen atom abstraction and O H addition to sulfur also holds for higher pH. Moreover, spectral evidence from peptides with methionine at the carboxylic termhalls suggests that an intramolecular three-electron bond between nitrogen and sulfur is effectively inhibited if these two atoms are separated by a peptide bond. Thus, the yield of the S.-.N-bonded species is evaluated to 40% of G ( 0 H ) = 5.6.30 The optical absorption at 390 nm in spectrum b amounts to Gt = 1.48 X lo4 dm3 mo1-I cm-I, which with G (111) 2.25 yields t390 6600 dm3 mol-' cm-I. Although the absorption of the S.m.0-bonded radicals (IV) is not clearly
=
+
H3N-CH-CO-NH-CH I /\ H27
H27
7=O
S I H3C
(IV)
distinguishable from that of (111), as S.-.O and S:.N bonds generally absorb in the same r e g i ~ n , 'we ~ - assume ~~ that the absorption at 390 nm represents solely the species (111) (see sections (31) Hiller, K.-0.; Asmus, K.-D. Int. J . Radiat. Biol. 1981, 40, 597. (32) BonifaEiE, M.; Miickel, H.; Bahnemann, D.; Asmus, K.-D. J . Chem. Soc., Perkin Trans. 2 1975, 675. (33) Mahling, S.;Asmus, K.-D.; Glass, R. S.;Hojjatie, M.; Wilson, G . S. J . Org. Chem. 1987, 52, 3717.
6384
The Journal of Physical Chemistry, Vol. 93, No. 17. 1989
Bobrowski and Holcman
TABLE I: Kinetic and Equilibrium Data of Transients Resulting from the Reaction of OH with Methionyl Peptides kOH
peptide L-Met-L-Met-L-Met L-Met-Gly-L-Met L-Met-L-Ala-L-Met L- Met-Gly-L-Met-L-Met
+ peptide,
dm' mol-' s? 1.0 1.1 1.0 1.2
x x x x
10'0 10'0 1010 10'0
k(S..O)
-
7.7 0.8 1.2 2.3
k(S:.O) (S.'.S)+9
x 104 x 104
x 104 x 104
C and D concerning pH formation profiles and the S:.O-S:.S interconversion). The S:.O-bonded intermediate (IV) of LMet-L-Met was not detected; this is most likely due to its fast decarboxylation or a fast deprotonation of its immediate precursor, i.e., monomeric sulfur-centered radical cation (see below). A significantly different transient absorption spectrum was obtained for the oxidation of L-Met-L-Met at pH 2.9. A broad, flat absorption is seen in the range of 300-600 nm, 4 ps after the pulse (curve c, Figure 1). It may be anticipated (and will be supported by the experimental findings described below) that this spectrum consists of a superposition of the 390-nm absorption of the S:.N-bonded species and the 490-nm absorption of the S:. S-bonded species. Seventy-four microseconds after the pulse, the 490-nm band had practically disappeared (curve d, Figure l ) , leaving the 390-nm band with about half its initial intensity. When the optical densities at 390 and 490 nm, recorded immediately after the pulse and normalized to the actual G(0H) value, are plotted as a function of the pH (Figure 1, inset), two oppositely oriented, S-shaped curves are obtained. These results indicate that the formation of these two species is complementary and essentially reflects the mode of a proton-assisted dehydroxylation of the primary OH adduct at the sulfur in the methionine unit containing the amine group (reactions lb' and 3). The 490-nm curve represents the yield of the S:.S-bonded radical cation (I), while the 390-nm curve could be associated with S:.N-bonded species (111). However, in contrast to the O H methionine ~ y s t e m these , ~ respective pH formation profiles are remarkably different. In the acidic region the increase in optical density at 490 nm does not parallel the decrease at 390 nm (Figure 1, inset). Thus, the yield of the S:.S-bonded radical cation at pH 0-1 is roughly twice that expected from the complementary replacement of the S:.N species by the S:.S species. The halfvalues of the 390- and 490-nm curves are obtained at pH 2.7 and 2.0, respectively. The half-value of 2.0 (490-nm curve) is close to the pK = 2.2 of the methionine carboxyl group, and this is an indication that the ionization of the carboxyl group may be important for the formation and stabillity of the intramolecular S:.S-bonded radical cation (I) and S:.O-bonded radical (IV). At pH < pK(COOH), protonation of the primary S:.OH adduct on sulfur atom of the free carboxyl group containing methionine unit is provided by the external proton (reaction lb') rather than by the proton from the protonated carboxyl group. Consecutive water elimination results in formation of the sulfur-centered, -S'+-, radical cation (reaction lb') followed by formation of intramolecular S:.S-bonded radical cation. From the steric point of view, the sulfur-oxygen interaction may also be established via the -S'+- (reaction 1b'), followed by ring closure which results in the formation of the protonated S:.O-bonded radical (V) (reaction 4):
s-I
+ Ht,
dm' mol-' s-I 1.0 1.2 5.0 1.9
k(S:.S)t
-
(S.'.N)+r s-I
2.3 x 105 2.2 x 10s 1.7 x 105
x 108
x 107
x 107 X IO8
PK 4.15 3.05 4.15 5.10
the formation of the S:.O-bonded species (IV) by a mechanism analogous to the formation of S.-.N-bonded species, (111),14 is possible in the kinetic competition with the external protonation of the O H adduct (reaction 5).
I
I
I
(W Species IV, however, can undergo proton-assisted d i s s o ~ i a t i o n ~ ~ resulting in conversion into the S:.S-bonded radical cation (reaction 6 ) :
I
I
I
t
"'"\jaOl :. 6
s I
1
(1)
Thus the presence of species IV can be expected only at higher pH. At pH > pK(C0OH) electrostatic interaction between the positive charge at sulfur and the negative charge of the ionized carboxyl group can facilitate ring closure and subsequent formation of the S:.O-bonded species (IV). However, it has been reportedI4 for S-methylcysteine that, at pH where the carboxyl group is ionized, the C 0 2 elimination readily occurs via the S:.O-bonded intermediate H3C-S.'.
0
yo NH3
This very fast decarboxylation is particularly enhanced by the a-positioned amino group but is effectively hampered in acidic ~olution.'~ Recently, we have shown18that in solutions of X-L-Met peptides (X = Gly, Ser) the relative yield of intermolecular S:. S-bonded radical cations was a factor of 2.3 lower at pH 5.15 than at pH 1, which is interpreted as the fast decarboxylation of the S:.O-bonded species to a-amino-type radicals (VI). Analogously, a fast formation of the S:.O-bonded species (IV) followed by fast decarboxylation (reaction 7) competing with a protonation of (IV)
(V)
Formation of species V might occur since the oxygen atom is able to provide a free p-electron pair, even though the carboxyl group is protonated. However, at low pH (
