The reduction of Cbl(III) to Cbl(II) by glutathione
The GSH complex with Cbl(III), Cbl(III)GSH, is found in mammalian cells and is believed to be one of the major forms of vitamin B12 serving as a substrate for Cbl(III) reductase [12
]. These findings have been the impetus for extensive in vitro
studies characterizing the complex. It is a very stable 1:1 complex formed by reacting aquocobalamin with GSH [10
]. The detailed structure of this complex obtained by means of 1
C 2D-NMR and X-ray absorption confirm that GSH is coordinated to the cobalt atom via the cysteine sulfur atom [14
]. In we show the rapid formation of the Cbl(III)GSH complex. This complex is stable and does not undergo reduction. However, with a larger excess of GSH there is a slow reduction of the Cbl(III) to Cbl(II), as shown in . The Job plots obtained both from the visible spectra () and the EPR data () indicate that a second GSH molecule is required for Cbl reduction.
The requirement for a second GSH indicates that the tightly bound GSH is not involved in the reduction. As the X-ray absorption data [19
] indicates, the first GSH is docked in the –OH position of Cbl(III)OH attached to the Co(III) atom at the β axial site where it is protected by the phytyl chains stretching out providing a “basket” kind of arrangement (see the structure of Cbl(III), ). This GSH replaces the ligand (OH or water) at the β-axial site and may actually stabilize the Cbl(III) oxidation state inhibiting reduction [25
]. Reduction takes place only when a second GSH binds.
Where does the second GSH involved in the reduction () bind? It is unlikely that a second GSH binds directly to cobalt, with the first GSH tightly bound to the β axial site and the benzimidazole bound trans to the GSH on the other side of the corrin at the α axial site. With no available cobalt coordination site the most reasonable area that seems to permit the GSH to approach the metal center and facilitate the reduction would be the void space available in the 5,6-dimethyl benzimidazole ribonucleotide ligand cavity (). The kinetics of the slow reduction () can be explained by the requirement for the relatively large GSH to enter this restricted cavity and achieve the proper location and orientation necessary for the transfer of an electron from the thiol to Co(III). The first GSH already coordinated to Cbl(III) in the β-axial site may contribute to the reduction process by lowering the reduction potential.
The general spectral features of GSH reduced cobalamin shown in the EPR spectra of and are similar to earlier observations [26
]. In at 77K, with the excess of GSH required for reduction, the nitrogen superhyperfine lines originating from the bound benzimidazole are partially resolved in one of the species. The improved resolution of these lines relative to DTT reduced cobalamin () can be explained by GSH still bound in the β axial position when cobalamin is reduced. GSH at this site will decrease the intermolecular interactions reducing dipolar interactions, resulting in less broadening of the superhyperfine lines.
With GSH there is clear evidence from both X-band and Q-band EPR for an additional species even with a 10 fold excess of GSH (). A single species should have eight equally speced hyperfine lines that originate from the quadrupolar effect of the cobalt-59 (I=7/2) nucleus. The presence of two species is evident by the number of hyperfine lines and the unequal splitting between these hyperfine lines. In addition nitrogen superhyperfine splitting is observed for only some of these lines, while other lines are sharp indicating the absence of any nitrogen interactions. These two species are the base-on species with triplet superhyperfine due to 14N and the base-off species with sharp lines due to 59Co hyperfine lines with no 14N splitting. Using the Q-band EPR spectra, it was possible to simulate both spectral components () obtaining the EPR parameters (see above). The base-off species is stabilized when Co(III) is reduced to Co(II), by a trans effect on the base due to GSH still bound in the β axial site and by a reduction of the pH when excess GSH is added.
With a large excess of GSH () the relative intensity of the base-on
species is reduced and there is an increase in the base off
species, with sharp A‖
Co) lines not broadened by nitrogen superhyperfine splitting. However, at these high concentrations of added GSH the base-off complex with g
= 2.272 obtained at a 10 fold excess of GSH () is no longer detected. Instead there is an increase in g
to 2.39 caused by the coordination of a second GSH in the α-axial site originally occupied by benzimidazole.
The Thiyl Radical Associated with GSH Reduced Cobalamin
The basis for radical involvement in Cbl chemistry goes back to the original Schrauzer [18
] proposal that the reduction of Cbl by GSH involves the formation of thiyl radicals.
The involvement of these radicals in Cbl dependent enzymes has been extensively studied [28
In order to determine if any of the thiyl radical formed when GSH reduces Cbl stays associated with Cbl(II), we measured GSSG formed during the reaction. Interestingly, the oxidized GSSG accounted for only ~ 50% of the thiyl radicals formed during the reduction. The other 50% of the thiyl radicals must, therefore, be associated with reduced Cbl.
Two distinct species involving additional interactions with GSH are detected by EPR. These species are only detected in the base-off
configuration, which is stabilized during the reduction by GSH (see above). The g
= 2.39 line together with the sharp Co hyperfine lines are associated with the coordination of a sulfur in the α-axial position. These lines are formed when excess GSH binds to reduced Cbl (complex F, see , vide infra). If the GSH on the α-axial side is a thiyl radical, similar EPR paramaters can be observed if the sulfur remains >2.2 Å from the Co atom. A closer approach results in the exchange coupled species (g=2.19), which reflects the capture of a spin from the thiyl radical by Cbl(II), referred to as ‘internal spin trapping’.
Sequence of reduction of Cbl(III)OH with GSH.
In order to prove that the g=2.19 band is not part of the species with g
=2.39, we simulated the spectrum of the g=2.39 species. As shown in (bottom), this simulation fits all the lines in the reduced cobinamide spectrum except for the line with g= 2.19. Confirmation that the g=2.19 line is not part of the g
=2.39 species is also provided by differences between these two lines in saturation studies performed by increasing the power () or decreasing the temperature (). These studies clearly indicate that the g=2.39 line and the sharper A‖
lines are saturated before the g=2.19 line.
The relatively broad (15 gauss) line with a g-value of 2.19 is clearly not an isolated free radical, which should have a g value much closer to the g-value of 2.0023 for a free electron [30
]. Even for sulphur based radicals, which have higher g-values, the value should be between 2.03 and 2.09 for an isolated radical [31
]. Very large exchange coupling or dipolar coupling between the metal center and the thiyl radical can give rise to the broad g= 2.19 isotropic line. This suggestion is, however, ruled out by the predicted very small exchange coupling constant (vide infra
). Hence this large-intensity line is interpreted to represent the g
of an exchange coupled species involving “internal spin trapping” between base-free Cbl(II) and the coordinated thiyl radical. The g
for this species should be the average between that of Cbl(II)(GSH)2
with GSH bound in the α and β axial position and that of the free thiyl radical. The ligand field for two GSH molecules bound would result in g=2.39 (complex F, see vide infra) and the estimated g
~ 2.00 for a free thiyl radical can be based on the theoretical calculations and experimental data reported by van Gastel and coworkers [31
]. The average results in a value of 2.195 in good agreement with the observed intense line at 2.19. It should, however, be noted that the g
-value of 2.00 being used for the thiyl radical is only a rough estimate. Van Gastel and coworkers [31
] have cautioned that the largest of the anisotropic g- parameters viz.
, which decides the magnitude of gav
is very sensitive to the conformation of the cysteine, the environment of sulfur and polarity. This is because the EPR g-values are very sensitive to the energy difference between the singly occupied almost degenerate levels and one of the lone-pair orbitals, the ΔE being of the order of 1000 to 4000cm−1
The parallel component of the exchange coupled species would be in the 2.0–2.05 range taking an average of 2.0 for the gzz
for the cobalt species and 2.0 to 2.10 for the thiyl radical [31
]. Similarly, the parallel component will have its 59
Co hyperfine lines at half the A‖
value observed for the g=2.39 species i.e. ~50–60 ×10−4
at much reduced intensities in comparison to the amplitude of g
= 2.19. The simulated spectrum of this species with g‖
= the 2.00, g
= 2.19, A
= 1 ×10−4
is shown on the top of . At least one or two of the predicted lines are poorly resolved as weak lines with + signs in the experimental spectrum ().
The temperature dependence in the 77-20 K reveals that the Co(II)-thiyl species is ferromagnetically coupled. The intensity variation of this data was used to calculate a ‘J’ value, i.e. Heisenberg exchange coupling, [34
] of 2.45 ± 0.2 cm−1
. In our analysis, we could not detect any effects due to dipolar coupling. Confirmation of a triplet state due to the exchange-coupled species is frequently obtained by the observation of a half-field transition. However, the intensity of the band due to this forbidden transition is proportional to the zero-field splitting (2D) and cannot be detected for the small D-value expected for our complex
Support for the assignment of the g=2.19 complex as a Co(II) thiyl exchange coupled species can be drawn from two important works involving ribonucleotide triphosphate reductase: (i) A protein based thiyl radical has been proposed as an intermediate in this enzyme from Lactobacillus leichmannil
, which catalyzes adenosyl cobalamin-dependent reduction as well as the exchange of the 5 hydrogens of adenosylcobalamin with solvent [37
]. (ii) Modeling of this intermediate as a thiyl radical coupled to cobalamin(II) by electron-electron exchange and dipolar interaction yields a reasonable fit to both X- and Q-band spectra. The J-value calculated by Gerfen et al [33
] was |Jex
| > 0.45 cm−1
for the cysteine thiyl radical) detected in cobalamin(II) reacted ribonucleotide triphosphate reductase. This determination of the J value by simulation was in the same range as the 2.45 cm−
1(see above) that we calculated from the temperature dependence of the intensity, which indicates that both complexes are similar. They also observed a minimal amount of dipolar broadening.
The reduction pathway of Cbl(III)OH by the addition of GSH is summarized in . The GSH adduct of Cbl(III), already established earlier by UV-Vis experiments, is represented by A, in this Scheme. The formation of this complex, Cbl(III)GSH, requires one GSH molecule and involves the simple displacement of OH by GSH. The binding of a second GSH in the α-axial ligand cavity is represented by B. The second GSH interacts with the cobalt as represented by C resulting in the reduction of Cbl(III) to Cbl(II) forming a thiyl radical (D and D’). The thiyl radical can be released and undergoes dimerization leading to the formation of oxidized glutathione GS:SG and the base-on reduced complex D. As indicated by our analysis of the formation of GSSG, only~ 50% of the radical goes on to form GSSG and the remainder of the GS• radicals formed are still associated with Cbl. The EPR parameters with the base still coordinated (the base-on complex D with g=2.234) is not significantly affected by having the thiyl radical (complex D’) in the pocket or if some of the excess GSH enters this pocket (complex D’’).
Under conditions where the base is still coordinated, the evidence for the formation of thiyl radicals is, therefore, indirect. The released thiyl could not be spin-trapped using an external spin trap in this experiment, since the formation of GS:SG through the process of radical recombination as well as the internal spin trapping by Cbl(II) are faster than the reaction of GS• with an external spin trap. At the low temperatures used, the EPR spectrum of the thiyl in the ligand pocket is broadened and not detected (vide infra).
In the pathway with the thiyl radical released, the initial low levels of a base-off
complex with g
=2.272 (E) is observed (). This is a 5-coordinated complex without any ligand substituting for the displaced base in the α-axial position. The dissociation of the base in this complex is facilitated by a trans effect associated with GSH bound in the β-axial site and the release of protons during the reduction process that lowers the pH. The affinity of GSH for the α-axial site is much less than that of the initial GSH bound to the β-axial site. However, with a large excess of GSH a second GSH binds in the α-axial site resulting in a complex with g
=2.39 (complex F).
In the pathway with the thiyl in the ligand pocket, as soon as the base is released the thiyl radical will interact with Co(II). A transient species formed as the thiyl radical approaches Co(II) would have parameters similar to complex F with two thiols bound with g
~2.39. However, this species is only formed transiently, because the closer approach of the thiyl radical produces an exchange coupled complex E’ with g
=2.19. This complex is present whenever base-off
complexes are observed (, ,), but is most pronounced when the base is completely removed as in cobinamide () or by lowering the pH and protonating the base (). At high GSH levels it is also possible that the thiyl radical bound in the α-axial position (species E’) can be displaced by GSH producing complex F.
Comparison of Cbl reduction by GSH and DTT
DTT a smaller molecule with two terminal thiols reduces Cbl much more efficiently and rapidly than GSH (). From the kinetics and spectral differences between GSH and DTT reduced Cbl, important insights can be provided into the reduction of Cbl by thiols.
Although the EPR spectrum at 77K obtained with DTT () is similar to that obtained for GSH (& ), the nitrogen superhyperfine lines with DTT are more poorly resolved than for GSH. We have attributed the better resolution for GSH (see above) to the binding of the larger GSH in the β-axial position which reduces intramolecular dipolar broadening. The broadening of the hyperfine lines for DTT can, however, also be attributed to increased retention of the formed thiyl radicals in the ligand pocket.
Proof that thiyl radicals are associated with DTT reduced Cbl even when the base is bound is provided by , where we show that lowering the pH of the DTT reduced base-on
complex results on the formation of the exchange coupled species with g
=2.19 (complex E’). The simultaneous formation of a base-off complex with g
=2.39 (complex F) that involves the binding of two thiols instead of a g
=2.272 (complex E) also indicates that DTT is bound to the β-axial side of the corrin. Although, DTT binding to the β axial side without the interactions involving the glu and gly residues of GSH [22
] is expected to be weaker than the binding of GSH, this linkage to the corrin can explain a greater retention of DTT thiyl radicals than the 50% retention observed for GSH. This retention can result in broadening of the nitrogen hyperfine lines observed with DTT. The initial β-axial binding also facilitates the access of the second DTT thiol to the α-axial ligand pocket, explaining the rapid reduction with DTT.
The binding of one of the DTT thiols in the β-axial position that increases the retention of the thiyl radical in the pocket is also expected to restrain the approach of the thiyl radical to cobalt when the base is dissociated. This can result in complex F where one of the sulfur atoms is part of a thiyl radical that does not approach Co(II) close enough to form the exchange coupled complex (E’).This is supported by the power saturation studies (). Thus, for the GSH reaction () the 2.39 line (complex F), which does not involve thiyl radicals, is saturated before the 2.19 line (complex E’), which involves a thiyl radical and, therefore, has a shorter relaxation time. However, for DTT () the saturation of the 2.39 line is similar to the 2.19 line implying that the 2.39 line (complex F) also involves a thiyl radical. The resultant complex, nevertheless, has the properties of two sulfur axial ligands and has the same EPR parameters as the GSH complex F.
The dominant species when DTT reduces Cbi () is a complex with nitrogen hyperfine interactions. These nitrogen hyperfine interactions in the EPR spectrum demonstrate that in the absence of the benzamidazole group one of the phytyl groups containing a nitrogen such as the longer –CH2-CH2-CO-NH-CH2-CHOH-CH3 or one of the two –CH2-CH2-CO-NH2 (see ) swing back to provide axial coordination through nitrogen, presumably in the α-axial site.
Support for this bonding is provided by a careful analysis of the EPR spectra obtained, which indicates a difference in the hyperfine splitting from 14
N for the benzimidazole and phyytyl nitrogens. The 14
N hyperfine splitting is less resolved in for Cbi than in and , for the benzimidazole, attributed to a reduction in the hyperfine splitting from 22.3 G to 20.3 G that is due to weaker binding. Such differences in 14
N hyperfine splittings have been observed in a study of the coordination of hyperlong nitrogen ligands in a series of LCo(II)Cbi+
adducts where L represents a series of substituted pyridines with 14
N hyperfine splittings varying from 18.3 to 19.8G [27
These nitrogen hyperfine lines are eliminated with an excess of GSH () implying that these relatively weak interactions with the phytyl nitrogens inhibit the coordination of the DTT thiyl radicals, but not GSH or the GSH thiyl radicals () with Co(II). These results are consistent with constraints on the interaction of the DTT thiyl radical imposed by binding the same molecule to the β-axial site (see above). It is only at low pH where both the benzimadazole group and any phytyl nitrogens are protonated that in the DTT spectrum the g
= 2.19 is clearly observed and the g
=2.39 becomes the dominant band ().
Since the g
= 2.19 line (complex E’) requires that the thiyl radical interact strongly with the Co(II), complex E’ formed with DTT would have the interaction on the β-axial side further weakened. When an excess of GSH is added to DTT reduced Cbl ( & ) no additional thiyl radicals are formed. However, GSH, which has a much higher affinity for the β axial Co(II) will displace DTT from the β axial side of the corrin. For the g
=2.19 complex E’, this reaction will not affect the properties of the complex. However, complex F with a constrained thiyl radical (see above) will be converted into the g
=2.19 complex E’. The conversion of complex F to complex E’ is seen by reduction in the F/E’ ratio from ~12 for DTT () to ~8 in after an excess of GSH is added. Furthermore, the saturation experiment () indicates that after an excess of GSH is added saturation of the 2.39 line occurs at a lower power than the 2.19 line indicating that thiyl radicals are no longer associated with complex F.