Skip to main content
NCBI home page
Protein Science : A Publication of the Protein Society logoLink to Protein Science : A Publication of the Protein Society
. 2009 Nov 9;19(1):190–194. doi: 10.1002/pro.269

The conserved active site tryptophan of thioredoxin has no effect on its redox properties

Goedele Roos 1,2,3,4,*, Paul Geerlings 4, Joris Messens 1,2,3
PMCID: PMC2817855  PMID: 19902501

Abstract

In Staphylococcus aureus thioredoxin (Trx) it has been shown that mutation of the conserved active site tryptophan residue (Trp28) has a large effect on the protein stability, on the pKa of the nucleophilic cysteine and on the redox potential. Since these effects can either be due to the partially unfolding of the Trp28Ala mutant or to the absence of the indole side chain of Trp28 as possible interaction partner for the active site cysteines, the origin of the experimentally observed effects is not known and is beyond experimental approach. With theoretical pKa and density functional theory reactivity analysis on model systems where Trp28 has been replaced by an alanine within the structural environment of Trx it is shown that Trp28 does not affect the redox parameters of Trx. As such, the experimentally observed redox effects of the Trx W28A mutant might be due to structural changes induced by partial unfolding.

Keywords: Density-functional calculations, enzyme catalysis, thioredoxin, HSAB principle

Introduction

Thioredoxin (Trx), a ubiquitous reductase, is one of the most important regulators of the cellular redox balance. The key biological activities of Trx are the promotion of cell growth, the inhibition of apoptosis, the protection against oxidative stress and the modulation of inflammation.1 All Trx feature a conserved active-site loop with a Trp-Cys-Gly-Pro-Cys2–4 sequence motif, numbered as Trp28 to Cys32 in Staphylococcus aureus Trx (Trx). In the structure of S. aureus Trx (2O7K),5 the indole side chain of Trp28 is turned towards the interior of the protein and covers an important part of the active side. There are no direct hydrogen bond interactions of Trp28 with the catalytic active cysteine residues. Mutation of the conserved active site tryptophan to alanine converts Trx into a domain swapped dimmer (3DIE).6 From the thermodynamic stability curves it was shown that both the reduced and oxidized monomeric forms of Trx W28A were severely affected by the mutation.6 They seem to be partially unfolded, reminiscent to a molten globule-like population.6 We previously showed that the pKa of Cys29 increases from 7.1 in wild type Trx to 8.3 in the monomeric form of Trx W28A,5 which is a pKa value more similar to the pKa of a cysteine out of its structural environment. Further, in Trx W28A, the redox potential decreases from −268 mV to −284 mV.5 It is not known whether these effects are due to conformational changes induced by the partially unfolding of the Trx W28A mutant or due to the absence of the conserved tryptophan indole side chain as possible interacting partner for the nucleophilic cysteines in the active site. It is not possible to exclude one of the possibilities experimentally, because upon the introduction of the Trp28Ala mutation, Trx shows the tendency to partially unfold.5 Therefore, theoretical pKa calculations and density functional theory (DFT)7–9 reactivity analysis on model systems with Trp28 replaced by alanine within the structural environment of wild type Trx are used to give insight in the intrinsic role of Trp28 on the redox behavior of Trx.

To explore and quantify the effect of Trp28 on the pKa of Cys29, the linear relationship between the calculated natural population analysis (NPA)10 charge on the thiol sulfur atoms and the pKa,11 is being used. The more negative the NPA charge on the sulfur atom, the higher the tendency to bind a proton, and the higher the thiol pKa. We have been able to successfully use the NPA-pKa correlation to determine the origin of the pKa perturbation of N-terminal cysteines in α- and 310-helices,12 in the study of the activation of the nucleophilic thiolates in the reaction catalyzed by arsenate reductase (ArsC)13 and during mixed disulfide complex dissociation of ArsC and Trx.11

In the conserved active site sequence motif Trp28-Cys29-Gly30-Pro31-Cys32 of Trx, we have studied the reactivity of Cys29 and Cys32 towards oxidized ArsC,14,15 an endogenous substrate of Trx. Trx reduces oxidized ArsC via the formation of a mixed Trx-ArsC disulfide complex16 (Fig. 1). To compare the reactivity of Cys29 and Cys32 in the presence and absence of the indole side chain of Trp28, we used the local version of the soft acids and bases principle (HSAB).17–19 This principle has been formulated in a conceptual DFT9 context and states that hard acids prefer to react with hard bases whereas soft acids prefer to interact with soft bases. Disulfide exchange reactions are soft-soft interactions; as such the softness is used as a reactivity descriptor. The smaller the difference in the local softness (s) between the sulfur atoms of the attacking nucleophilic cysteines and the accepting electrophilic Cys82ArsC-Cys89ArsC disulfide of oxidized ArsC (Fig. 1), the more preferred the interaction is:

graphic file with name pro0019-0190-m1.jpg (1)

with s+ and s− the local softness of the electrophilic and the nucleophilic sulfur atoms, here the sulfur atoms of the cysteine residues in respectively the Cys82ArsC-Cys89ArsC disulfide (Fig. 1), and Cys29Trx and Cys32Trx.

Figure 1.

Figure 1

Open in a new tab

Trx reduces oxidized ArsC via an intermediate Trx-ArsC complex: (A) The reaction takes of with a nucleophilic attack of Cys29Trx of reduced Trx on Cys89ArsC of the Cys82ArsC-Cys89ArsC disulfide in oxidized ArsC (1Z2E), leading to the formation of the Cys29Trx-Cys89ArsC mixed disulfide (2IPA). (B) In a second step, Cys32Trx attacks Cys29Trx, leading to the release of reduced ArsC (1Z2D) and oxidized Trx (2GZZ) (C). The figure was generated by using PyMol v099 (Delano Scientific LLC, 2006).

The local softness s is is obtained as

graphic file with name pro0019-0190-m2.jpg (2)
graphic file with name pro0019-0190-m3.jpg (3)

in which S is the global softness, a global property that correlates with the system's polarizability.9 f is the Fukui function8,9 and indicates the orientation effect of a chemical reaction: which nucleophilic sulfur atom (f−) will attack and which sulfur (f+) of the disulfide will receive electrons. The − and + sign indicate the reactivity towards nucleophilic and electrophlic attacks respectively. f− is related to the electron density of the highest occupied molecular orbital and f+ to the density of the lowest unoccupied molecular orbital when electrons are received.20 The global and local softness and the Fukui function are well established and extensively used to study generalized acid/base reactions including most of the organic reactions (additions, substitutions, eliminations)9 of which disulfide exchange reactions are an example.

In sum, the local softness descriptors are combined with pKa calculations to give fresh insights in the experimentally observed effects of the Trp28Ala mutation in Trx.

Results and Discussion

To get insight into the origin of the experimentally observed effects of the Trp28Ala mutation in Trx, we have performed pKa calculations and reactivity analysis on a model system of reduced Trx in which Trp28 is replaced by alanine.

The pKa of Cys29Trx in wild type reduced Trx was calculated to be 5.5.11 By replacing Trp28 by alanine within the structural environment of wild type reduced Trx (Trx_red_C29 model in Table I), the pKa does not change. Further, in this model, the removal of the indole side chain of Trp28 does not alter the local softness difference between the sulfur atoms of Cys29Trx and the sulfur atoms of the Cys82ArsC-Cys89ArsC disulfide, and thus, no effect on the reactivity of Trx towards ArsC (Table II) has been observed. Both, the Fukui function and the global softness of Cys29Trx and Cys32Trx in the Trx W28A model are similar to those found in the model of wild type Trx (Table II). In the Trx W28A model, the sulfur atoms of Cys29Trx and Cys32Trx have comparable Fukui function values, corresponding to the same intrinsic reactivity. Cys29Trx has a lower global softness than Cys32Trx, explaining its higher reactivity towards the less soft Cys82ArsC-Cys89ArsC disulfide (Table II). As such, in Trx W28A the experimentally observed regioselectivity of Cys29Trx to nucleophilically attack Cys82ArsC of the disulfide in oxidized ArsC is directed by the difference in global softness between Cys29Trx and Cys32Trx. These results are analogous to the calculations with a model of wild type Trx, and thus within the structural environment of wild type Trx, Trp28 has no effect on reactivity, regioselectivity and on the pKa of Cys29Trx. As such, the experimentally obtained results5,6 of an increased pKa of Cys29Trx and a decreased redox potential in Trx W28A most likely originates form the structural effects induced by the partial unfolding of Trx W28A.

Table I.

Trp28 has no Effect on the pKa of Cys29 and Cys32 in Reduced Trx and in the Trx-ArsC Complex

Model system PDB Cysteine residue qNPA pKa
Trx Trx_red_C29 2GZY C29Trx −0.671 5.511
Trx_red_C29 W28A 2GZY C29Trx −0.673 5.5
Trx-ArsC Trx_ArsC_C32 2IPA C32Trx −0.791 8.311
Trx_ArsC_C32 W28A 2IPA C32Trx −0.791 8.3
Open in a new tab

Calculated pKa's of the nucleophilic cysteines of Trx and in the Trx-ArsC complex. The pKa values are obtained via the NPA-pKa correlation.11

Table II.

Trp28 has no Effect on the Reactivity of Reduced Trx

Model system Cysteine residue S f+/f− s+/s−
Trx (2GZY)  Trx_red_C29 C29Trx 6.19a 0.866a 5.36a
 Trx_red_C32 C32Trx 8.68a 0.858a 7.45a
Trx (2GZY)  Trx_red_C29 W28A C29Trx 6.06 0.866 5.25
 Trx_red_C32 W28A C32Trx 8.73 0.859 7.50
Δs C29Trx-C82ArsC C29Trx-C89ArsC C32Trx-C82ArsC C32Trx-C89ArsC
Trx_red – ArsC_ox 4.73a 4.58a 6.82a 6.66a
Trx_red W28A – ArsC_ox 4.62 4.47 6.87 6.71
Open in a new tab
a

Values obtained from Roos et al.11 Global softness (S), Fukui function (f+ or f−) and local softness (s+ or s−) values of the nucleophilic cysteines in Trx. Reactivity of the sulfur atoms of Cys29Trx and Cys32Trx of Trx towards the sulfur atoms of the Cys82ArsC-Cys89ArsC disulfide of ArsC as measured by the difference in local softness [Δs, Eq. (1)].

Also in the Trx-ArsC complex (Fig. 1), Trp28 has no effect on the pKa and the reactivity. The pKa of Cys32Trx in the model of wild type Trx-ArsC is calculated to be 8.311 and does not change upon replacing Trp28 by alanine within the structural environment of the complex between wild type Trx and ArsC (Trx_ArsC_C32 model in Table I). Further, in this model, the local softness difference between the sulfur atoms of Cys32Trx and Cys29Trx is similar to this in the complex between wild type Trx and ArsC (Table III). Both, the Fukui function and the global softness of Cys32Trx and of Cys29Trx in the Trx-ArsC W28A model do not differ significantly from the values found in the wild type Trx-ArsC model.

Table III.

Trp28 has no Effect on the Reactivity of Trx in the Trx-ArsC Complex

Trx-ArsC complex (2IPA) Cysteine residue S f+/f− s+/s−
Trx_ArsC_C32 C32Trx 6.47a 0.865a 5.59a
C29Trx 5.97a 0.522a 3.12a
C89ArsC 5.97a 0.122a 0.73a
Trx_ArsC_C32 W28A C32Trx 6.49 0.865 5.61
C29Trx 5.33 0.517 2.76
C89ArsC 5.33 0.162 0.86
Δs C32Trx-C29Trx C32Trx-C89ArsC C82ArsC-C29Trx
Trx_ArsC_C32 2.48a 4.86a 4.21a
Trx_ArsC_C32 W28A 2.85 4.75 4.59
Open in a new tab
a

Values obtained from Roos et al.11 Global softness (S), Fukui function (f+ or f−) and local softness (s+ or s−) values of the nucleophilic cysteines in Trx-ArsC complex. Reactivity of the sulfur atoms of Cys32Trx towards the Cys29Trx-Cys89ArsC mixed disulfide as measured by the difference in local softness [Δs, Eq. (1)].

All together, as pointed out in the Introduction section, it has been shown that Trp28 has a large effect on the protein structure, on the pKa of the nucleophilic cysteine and on the redox potential of Trx.5 Theoretical calculations performed on a model system in which the structural effects of partial unfolding are not present indicate that Trp28 has no influence on the pKa of the nucleophilic cysteine in neither reduced Trx or in the Trx-ArsC complex (Table I). Furthermore, the minimal softness criterion indicates that Trp28 has no effect on the reactivity and on the regioselectivity during Trx-ArsC complex formation (Table II) and dissociation (Table III). As such, the effects of Trp28 on the redox properties of Trx might be assigned to the partially unfolding of thioredoxin when the Trp28Ala mutation is introduced. Equally important we have demonstrated that theoretical calculations can help to rationalize experimental results and can give fresh insight in questions which are currently beyond experimental approach.

Model systems and Computational Details

Model systems of Bacillus subtilis Trx (Trx) and B. subtilis Trx-ArsC (Trx-ArsC) complex are built based on the structures with PDB code 2GZY and 2IPA. Reduced Trx (2GZY)21 is modeled by the W28-C32 active site and the adjacent α1-helix (K33-E45). In Trx_red_C29, C29 is deprotonated and in Trx_red_C32, both C29 and C32 are deprotonated. The Trx_ArsC complex (2IPA)22 is modeled (Trx-ArsC_C32) by the Trx active site (W28Trx-C32Trx) and the ArsC redox helix (C82ArsC-C89ArsC) and T11ArsC and R16ArsC; C32Trx and C82ArsC are deprotonated The W28A mutation is built in silico starting from the coordinates of Trx_red_C29, Trx_ArsC_C32. In all models, hydrogen atoms are placed and optimized together with the Sγ atoms of the reduced cysteine residues at the B3LYP/6-31G* level. NPA charges, Fukui functions and softnesses are calculated as described.11 All calculations were performed with the Gaussian 03 package.23

Acknowledgments

G. R. thank the Fund for Scientific Research (FWO) for a postdoctoral fellowship. P. G. thank the VUB and the FWO for continuous support to his group. J. M. is a project leader of the VIB.

References

  • 1.Burke-Gaffney A, Callister ME, Nakamura H. Thioredoxfriend or foe in human disease? Trends Pharmacol Sci. 2005;26:398–404. doi: 10.1016/j.tips.2005.06.005. [DOI] [PubMed] [Google Scholar]
  • 2.Messens J, Van Molle I, Vanhaesebrouck P, Limbourg M, Van Belle K, Wahni K, Martins JC, Loris R, Wyns L. How thioredoxin can reduce a buried disulphide bond. J Mol Biol. 2004;339:527–537. doi: 10.1016/j.jmb.2004.04.016. [DOI] [PubMed] [Google Scholar]
  • 3.Aslund F, Berndt KD, Holmgren A. Redox potentials of glutaredoxins and other thiol-disulfide oxidoreductases of the thioredoxin superfamily determined by direct protein-protein redox equilibria. J Biol Chem. 1997;272:30780–30786. doi: 10.1074/jbc.272.49.30780. [DOI] [PubMed] [Google Scholar]
  • 4.Eklund H, Gleason FK, Holmgren A. Structural and functional relations among thioredoxins of different species. Proteins. 1991;11:13–28. doi: 10.1002/prot.340110103. [DOI] [PubMed] [Google Scholar]
  • 5.Roos G, Garcia Pino A, Van Belle K, Brosens E, Wahni K, Vandenbussche G, Wyns L, Loris R, Messens J. The conserved active site proline determines the reducing power of Staphylococcus aureus thioredoxin. J Mol Biol. 2007;368:800–811. doi: 10.1016/j.jmb.2007.02.045. [DOI] [PubMed] [Google Scholar]
  • 6.Garcia-Pino A, Martinez-Rodriguez S, Wahni K, Wyns L, Loris R, Messens J. Coupling of domain swapping to kinetic stability in a thioredoxin mutant. J Mol Biol. 2009;358:1590–1599. doi: 10.1016/j.jmb.2008.11.040. [DOI] [PubMed] [Google Scholar]
  • 7.Hohenberg P, Kohn W. Inhomogeneous electron gas. Phys Rev. 1960;136:B864–B871. [Google Scholar]
  • 8.Parr RG, Yang W. Density-functional theory of atoms and molecules. New York: Oxford University Press; 1989. [Google Scholar]
  • 9.Geerlings P, De Proft F, Langenaeker W. Conceptual density functional theory. Chem Rev. 2003;103:1793–1873. doi: 10.1021/cr990029p. [DOI] [PubMed] [Google Scholar]
  • 10.Reed AE, Curtiss LA, Weinhold F. Intermolecular interactions from a natural bond orbital, donor-acceptor viewpoint. Chem Rev. 1988;88:899–926. [Google Scholar]
  • 11.Roos G, Foloppe N, Van Laer K, Wyns L, Nilsson L, Geerlings P, Messens J. How thioredoxin dissociates its mixed disulfide. Plos Comp Biol. 2009;5:e1000461. doi: 10.1371/journal.pcbi.1000461. doi: 10.1371/journal.pcbi.1000461. [DOI] [PMC free article] [PubMed] [Google Scholar]
  • 12.Roos G, Loverix S, Geerlings P. Origin of the pK(a) perturbation of N-terminal cysteine in alpha- and 3(10)-helices: a computational DFT study. J Phys Chem B. 2006;110:557–562. doi: 10.1021/jp0549780. [DOI] [PubMed] [Google Scholar]
  • 13.Roos G, Loverix S, Brosens E, Van Belle K, Wyns L, Geerlings P, Messens J. The activation of electrophile, nucleophile and leaving group during the reaction catalysed by pI258 arsenate reductase. Chembiochem. 2006;7:981–989. doi: 10.1002/cbic.200500507. [DOI] [PubMed] [Google Scholar]
  • 14.Ji G, Garber EA, Armes LG, Chen CM, Fuchs JA, Silver S. Arsenate reductase of Staphylococcus aureus plasmid pI258. Biochemistry. 1994;33:7294–7299. doi: 10.1021/bi00189a034. [DOI] [PubMed] [Google Scholar]
  • 15.Messens J, Silver S. Arsenate reduction: thiol cascade chemistry with convergent evolution. J Mol Biol. 2006;362:1–17. doi: 10.1016/j.jmb.2006.07.002. [DOI] [PubMed] [Google Scholar]
  • 16.Guo X, Li Y, Peng K, Hu Y, Li C, Xia B, Jin C. Solution structures and backbone dynamics of arsenate reductase from Bacillus subtilis: reversible conformational switch associated with arsenate reduction. J Biol Chem. 2005;280:39601–39608. doi: 10.1074/jbc.M508132200. [DOI] [PubMed] [Google Scholar]
  • 17.Pearson RG. Chemical hardness. Weinheim: Wiley-VCH; 1997. [Google Scholar]
  • 18.Gázquez JL, Mendez F. The hard and soft acids and bases principle: an atoms in molecules viewpoint. J Phys Chem. 1994;98:4691–4593. [Google Scholar]
  • 19.Damoun S, Van Dewoude G, Mendez F, Geerlings P. Local softness as a regioselectivity indicator in [4+2] cycloaddition reactions. J Phys Chem A. 1997;101:886–893. [Google Scholar]
  • 20.Yang W, Parr RG, Pucci R. Electron density, Kohn–Sham frontier orbitals, and Fukui functions. J Chem Phys. 1984;81:2862. [Google Scholar]
  • 21.Xu H. The solution structure of Bacillus subtilis thioredoxin in the reduced and oxidized forms. (in press) [Google Scholar]
  • 22.Li Y, Hu Y, Zhang X, Xu H, Lescop E, Xia B, Jin C. Conformational fluctuations coupled to the thiol-disulfide transfer between thioredoxin and arsenate reductase in Bacillus subtilis. J Biol Chem. 2007;282:11078–11083. doi: 10.1074/jbc.M700970200. [DOI] [PubMed] [Google Scholar]
  • 23.Frisch MJ. 2003. Gaussian 03, Revision A.1. Pittsburgh: Gaussian, Inc.

Articles from Protein Science : A Publication of the Protein Society are provided here courtesy of The Protein Society

RESOURCES