Many cellular functions involve cysteine chemistry via thiol–disulfide exchange pathways. The nucleophilic cysteines of the enzymes involved are activated as thiolate. A thiolate is much more reactive than a neutral thiol. Therefore, determining and understanding the pKas of functional cysteines are important aspects of biochemistry and molecular biology with direct implications for redox signaling. Here, we describe the experimental and theoretical methods to determine cysteine pKa values, and we examine the factors that control these pKas. Drawing largely on experience gained with the thioredoxin superfamily, we examine the roles of solvation, charge–charge, helix macrodipole, and hydrogen bonding interactions as pKa-modulating factors. The contributions of these factors in influencing cysteine pKas and the associated chemistry, including the relevance for the reaction kinetics and thermodynamics, are discussed. This analysis highlights the critical role of direct hydrogen bonding to the cysteine sulfur as a key factor modulating the equilibrium between thiol S–H and thiolate S−. This role is easily understood intuitively and provides a framework for biochemical functional insights. Antioxid. Redox Signal. 18, 94–127.
AdmanE, WatenpaughKD, JensenLH. NH–S hydrogen bonds in Peptococcus aerogenes ferredoxin, Clostridium pasteurianum rubredoxin, and Chromatium high potential iron protein. Proc Natl Acad Sci U S A, 72:4854–4858. 1975.
2.
AlexovE, MehlerEL, BakerN, AMB, HuangY, MillettiF, Erik NielsenJ, FarrellD, CarstensenT, OlssonMH, ShenJK, WarwickerJ, WilliamsS, WordJM. Progress in the prediction of pK(a) values in proteins. Proteins, 79:3260–3275. 2011.
3.
AlexovEG, GunnerMR. Incorporating protein conformational flexibility into the calculation of pH-dependent protein properties. Biophys J, 74:2075–2093. 1997.
4.
AntosiewiczJ, McCammonJA, GilsonMK. Prediction of pH-dependent properties of proteins. J Mol Biol, 238:415–436. 1994.
5.
AntosiewiczJ, McCammonJA, GilsonMK. The determinants of pKas in proteins. Biochemistry, 35:7819–7833. 1996.
6.
ÅqvistJ, LueckeH, QuiochoFA, WarshelA. Dipoles localized at helix termini of proteins stabilize charges. Proc Natl Acad Sci U S A, 88:2026. 1991.
7.
ArmstrongRN. Structure, catalytic mechanism, and evolution of the glutathione transferases. Chem Res Toxicol, 10:2–18. 1997.
8.
AslundF, BerndtKD, HolmgrenA. Redox potentials of glutaredoxins and other thiol-disulfide oxidoreductases of the thioredoxin superfamily determined by direct protein-protein redox equilibria. J Biol Chem, 272:30780–30786. 1997.
9.
ÅslundF, EhnB, Miranda-VizueteA, PueyoC, HolmgrenA. Two additional glutaredoxins exist in Escherichia coli: glutaredoxin 3 is a hydrogen donor for ribonucleotide reductase in a thioredoxin/glutaredoxin 1 double mutant. Proc Natl Acad Sci U S A, 91:9813–9817. 1994.
10.
BacikJP, HazesB. Crystal structures of a poxviral glutaredoxin in the oxidized and reduced states show redox-correlated structural changes. J Mol Biol, 365:1545–1558. 2007.
11.
BarkayT, MillerSM, SummersAO. Bacterial mercury resistance from atoms to ecosystems. FEMS Microbiol Rev, 27:355–384. 2003.
12.
BasDC, RogersDM, JensenJH. Very fast prediction and rationalization of pKa values for protein-ligand complexes. Proteins, 73:765–783. 2008.
13.
BashfordD, KarplusM. pKa's of Ionizable groups in proteins: Atomic detail from a continuum electrostatic model. Biochemistry, 29:10219–10225. 1990.
14.
BeneschRE, BeneschR. The acid strength of the -SH Group in cysteine and related compounds. J Am Chem Soc, 77:5877–5881. 1955.
15.
BessettePH, CottoJJ, GilbertHF, GeorgiouG. In vivo and in vitro function of the Escherichia coli periplasmic cysteine oxidoreductase DsbG. J Biol Chem, 274:7784–7792. 1999.
16.
BillietL, GeerlingsP, MessensJ, RoosG. The thermodynamics of thiol sulfenylation. Free Radic Biol Med, 52:1473–1485. 2012.
17.
BulajG, KortemmeT, GoldenbergDP. Ionization-reactivity relationships for cysteine thiols in polypeptides. Biochemistry, 37:8965–8972. 1998.
18.
BurleySK, PetskoGA. Aromatic-aromatic interaction: a mechanism of protein structure stabilization. Science, 229:23–28. 1985.
19.
CarvalhoAP, FernandesPA, RamosMJ. Similarities and differences in the thioredoxin superfamily. Prog Biophys Mol Biol, 91:229–248. 2006.
20.
ChenWJ, GraminskiGF, ArmstrongRN. Dissection of the catalytic mechanism of isozyme 4–4 of glutathione S-transferase with alternative substrates. Biochemistry, 27:647–654. 1988.
21.
ChengZ, ArscottLD, BallouDP, WilliamsCHJr.The relationship of the redox potentials of thioredoxin and thioredoxin reductase from Drosophila melanogaster to the enzymatic mechanism: reduced thioredoxin is the reductant of glutathione in Drosophila. Biochemistry, 46:7875–7885. 2007.
22.
ChengZ, ZhangJ, BallouDP, WilliamsCHJr.Reactivity of thioredoxin as a protein thiol-disulfide oxidoreductase. Chem Rev, 111:5768–5783. 2011.
23.
ClickTH, KaminskiGA. Reproducing basic pKa values for turkey ovomucoid third domain using a polarizable force field. J Phys Chem B, 113:7844–7850. 2009.
24.
ColletJF, MessensJ. Structure, function, and mechanism of thioredoxin proteins. Antioxid Redox Signal, 13:1205–1216. 2010.
25.
ColletJF, RiemerJ, BaderMW, BardwellJC. Reconstitution of a disulfide isomerization system. J Biol Chem, 277:26886–26892. 2002.
26.
CRC. Handbook of Chemistry and Physics. Florida: CRC Press, 1994.
27.
CreightonTE. Proteins: Structures and Molecular Properties. New York: W.H. Freeman, 1993.
28.
CrowA, AchesonRM, Le BrunNE, OubrieA. Structural basis of Redox-coupled protein substrate selection by the cytochrome c biosynthesis protein ResA. J Biol Chem, 279:23654–23660. 2004.
29.
D'AmbrosioK, PedoneE, LangellaE, De SimoneG, RossiM, PedoneC, BartolucciS. A novel member of the protein disulfide oxidoreductase family from Aeropyrum pernix K1: structure, function and electrostatics. J Mol Biol, 362:743–752. 2006.
DemchukE, WadeRC. Improving the continuum dielectric approach to calculating pKas of ionizable groups in proteins. J Phys Chem, 100:17373–17387. 1996.
32.
DepuydtM, MessensJ, ColletJF. How proteins form disulfide bonds. Antioxid Redox Signal, 15:49–66. 2011.
33.
DesirajuGR, SteinerT. IUCr Monographs on Crystallography, 9. Oxford: Oxford University Press/International Union of Crystallography, 1999.
34.
DickhoutJG, CarlisleRE, AustinRC. Interrelationship between cardiac hypertrophy, heart failure, and chronic kidney disease: endoplasmic reticulum stress as a mediator of pathogenesis. Circ Res, 108:629–642. 2011.
35.
DilletV, DysonHJ, BashfordD. Calculations of electrostatic interactions and pKas in the active site of Escherichia coli thioredoxin. Biochemistry, 37:10298–10306. 1998.
36.
DiscolaKF, de OliveiraMA, Rosa CussiolJR, MonteiroG, BarcenaJA, PorrasP, PadillaCA, GuimaraesBG, NettoLE. Structural aspects of the distinct biochemical properties of glutaredoxin 1 and glutaredoxin 2 from Saccharomyces cerevisiae. J Mol Biol, 385:889–901. 2009.
37.
DonohueJ. On N-H–S hydrogen bonds. J Mol Biol, 45:231–235. 1969.
38.
DysonHJ, JengMF, TennantLL, SlabyI, LindellM, CuiDS, KuprinS, HolmgrenA. Effects of buried charged groups on cysteine thiol ionization and reactivity in Escherichia coli thioredoxin: structural and functional characterization of mutants of Asp 26 and Lys 57. Biochemistry, 36:2622–2636. 1997.
39.
DysonHJ, TennantLL, HolmgrenA. Proton-transfer effects in the active-site region of Escherichia coli thioredoxin using two-dimensional 1H NMR. Biochemistry, 30:4262–4268. 1991.
40.
El HajjajiH, DumoulinM, MatagneA, ColauD, RoosG, MessensJ, ColletJF. The zinc center influences the redox and thermodynamic properties of Escherichia coli thioredoxin 2. J Mol Biol, 386:60–71. 2009.
41.
Ferrer-SuetaG, MantaB, BottiH, RadiR, TrujilloM, DenicolaA. Factors affecting protein thiol reactivity and specificity in peroxide reduction. Chem Res Toxicol, 24:434–450. 2011.
FoloppeN, NilssonL. The glutaredoxin -C-P-Y-C- motif: influence of peripheral residues. Structure, 12:289–300. 2004.
44.
FoloppeN, NilssonL. Stabilization of the catalytic thiolate in a mammalian glutaredoxin: structure, dynamics and electrostatics of reduced pig glutaredoxin and its mutants. J Mol Biol, 372:798–816. 2007.
45.
FoloppeN, SagemarkJ, NordstrandK, BerndtKD, NilssonL. Structure, dynamics and electrostatics of the active site of glutaredoxin 3 from Escherichia coli: Comparison with functionally related proteins. J Mol Biol, 310:449–470. 2001.
46.
Forman-KayJD, CloreGM, GronenbornAM. Relationship between electrostatics and redox function in human thioredoxin: characterization of pH titration shifts using two-dimensional homo- and heteronuclear NMR. Biochemistry, 31:3442–3452. 1992.
47.
GanZ, WellsWW. Identification and reactivity of the catalytic site of pig liver thioltransferase. J Biol Chem, 262:6704–6797. 1987.
48.
GanZ-R, SardanaMK, JacobsJW, PolokoffMA. Yeast thioltransferase-the active site cysteines display differential reactivity. Arch Biochem Biophys, 282:110–115. 1990.
49.
GanePJ, FreedmanRB, WarwickerJ. A molecular model for the redox potential difference between thioredoxin and DsbA, based on electrostatics calculations. J Mol Biol, 249:376–387. 1995.
50.
This reference has been deleted.
51.
GeorgescuRE, AlexovEG, GunnerMR. Combining conformational flexibility and continuum electrostatics for calculating pK(a)s in proteins. Biophys J, 83:1731–1748. 2002.
52.
GilbertHF. Molecular and cellular aspects of thiol-disulfide exchange. Adv Enzymol Relat Areas Mol Biol, 63:69–172. 1990.
53.
GilsonMK. Multiple-site titration and molecular modeling: two rapid methods for computing energies and forces for ionizable groups in proteins. Proteins, 15:266–282. 1993.
54.
GilsonMK, HonigB. Calculation of the total electrostatic energy of a macromolecular system: solvation energies, binding energies, and conformational analysis. Proteins, 4:7–18. 1988.
55.
GilsonMK, HonigBH. The dielectric constant of a folded protein. Biopolymers, 25:2097–2119. 1986.
56.
GrauschopfU, WintherJR, KorberP, ZanderT, DallingerP, BardwellJC. Why is DsbA such an oxidizing disulfide catalyst?Cell, 83:947–955. 1995.
GriffithsSW, KingJ, CooneyCL. The reactivity and oxidation pathway of cysteine 232 in recombinant human alpha 1-antitrypsin. J Biol Chem, 277:25486–25492. 2002.
59.
GuY, KarT, ScheinerS. Fundamental Properties of the C-H···O Interaction: Is it a True Hydrogen Bond?J Am Chem Soc, 121:9411–9422. 1999.
60.
GuY, SinghSV, JiX. Residue R216 and catalytic efficiency of a murine class alpha glutathione S-transferase toward benzo[a]pyrene 7(R),8(S)-diol 9(S), 10(R)-epoxide. Biochemistry, 39:12552–12557. 2000.
61.
GuddatLW, BardwellJC, GlockshuberR, Huber-WunderlichM, ZanderT, MartinJL. Structural analysis of three His32 mutants of DsbA: support for an electrostatic role of His32 in DsbA stability. Protein Sci, 6:1893–1900. 1997.
62.
HarrisTK, TurnerGJ. Structural basis of perturbed pKa values of catalytic groups in enzyme active sites. IUBMB Life, 53:85–98. 2002.
63.
HatahetF, RuddockLW. Protein disulfide isomerase: a critical evaluation of its function in disulfide bond formation. Antioxid Redox Signal, 11:2807–2850. 2009.
64.
HawkinsHC, FreedmanRB. The reactivities and ionization properties of the active-site dithiol groups of mammalian protein disulphide-isomerase. Biochem J, 275,Pt 2:335–339. 1991.
HenneckeJ, SpleissC, GlockshuberR. Influence of acidic residues and the kink in the active-site helix on the properties of the disulfide oxidoreductase DsbA. J Biol Chem, 272:189–195. 1997.
67.
HolWG, van DuijnenPT, BerendsenHJ. The alpha-helix dipole and the properties of proteins. Nature, 273:443–446. 1978.
68.
HonigB, NichollsA. Classical electrostatics in biology and chemistry. Science, 268:1144–1149. 1995.
69.
Huber-WunderlichM, GlockshuberR. A single dipeptide sequence modulates the redox properties of a whole enzyme family. Fold Des, 3:161–171. 1998.
70.
HugoM, TurellL, MantaB, BottiH, MonteiroG, NettoLE, AlvarezB, RadiR, TrujilloM. Thiol and sulfenic acid oxidation of AhpE, the one-cysteine peroxiredoxin from Mycobacterium tuberculosis: kinetics, acidity constants, and conformational dynamics. Biochemistry, 48:9416–9426. 2009.
71.
HunterCA, SandersJKM. The Nature of π−π Interactions. J Am Chem Soc, 112:5525–5534. 1990.
72.
IngelmanM, NordlundP, EklundH. The structure of a reduced mutant T4 glutaredoxin. FEBS Lett, 370:209–211. 1995.
73.
IqbalsyahTM, MoutevelisE, WarwickerJ, ErringtonN, DoigAJ. The CXXC motif at the N terminus of an α-helical peptide. Protein Sci, 15:1945–1950. 2006.
74.
JacobiA, Huber-WunderlichM, HenneckeJ, GlockshuberR. Elimination of all charged residues in the vicinity of the active-site helix of the disulfide oxidoreductase DsbA. Influence of electrostatic interactions on stability and redox properties. J Biol Chem, 272:21692–21699. 1997.
75.
JaoSC, English OspinaSM, BerdisAJ, StarkeDW, PostCB, MieyalJJ. Computational and mutational analysis of human glutaredoxin (thioltransferase): probing the molecular basis of the low pKa of cysteine 22 and its role in catalysis. Biochemistry, 45:4785–4796. 2006.
76.
JengMF, CampbellAP, BegleyT, HolmgrenA, CaseDA, WrightPE, DysonHJ. High-resolution solution structures of oxidized and reduced Escherichia coli thioredoxin. Structure, 2:853–868. 1994.
77.
JengMF, HolmgrenA, DysonHJ. Proton sharing between cysteine thiols in Escherichia coli thioredoxin: implications for the mechanism of protein disulfide reduction. Biochemistry, 34:10101–10105. 1995.
78.
JensenKS, HansenRE, WintherJR. Kinetic and thermodynamic aspects of cellular thiol-disulfide redox regulation. Antioxid Redox Signal, 11:1047–1058. 2009.
79.
JiX, von RosenvingeEC, JohnsonWW, TomarevSI, PiatigorskyJ, ArmstrongRN, GillilandGL. Three-dimensional structure, catalytic properties, and evolution of a sigma class glutathione transferase from squid, a progenitor of the lens S-crystallins of cephalopods. Biochemistry, 34:5317–5328. 1995.
80.
JonesDP, GoYM. Mapping the cysteine proteome: analysis of redox-sensing thiols. Curr Opin Chem Biol, 15:103–112. 2011.
81.
JoshiHV, MeierMS. The effect of a peptide helix macrodipole on the pKa of an Asp side chain carboxylate. J Am Chem Soc, 118:12038–12044. 1996.
82.
KallisGB, HolmgrenA. Differential reactivity of the functional sulfhydryl groups of cysteine-32 and cysteine-35 present in the reduced form of thioredoxin from Escherichia coli. J Biol Chem, 255:10261–10265. 1980.
83.
KaralaAR, LappiAK, RuddockLW. Modulation of an active-site cysteine pKa allows PDI to act as a catalyst of both disulfide bond formation and isomerization. J Mol Biol, 396:883–892. 2010.
84.
KerrKA, AshmoreJP, KoetzleTF. A neutron diffraction study of L-cysteine. Acta Cryst, B31:2022–2026. 1975.
KhareD, AlexanderP, AntosiewiczJ, BryanP, GilsonM, OrbanJ. pKa measurements from nuclear magnetic resonance for the B1 and B2 immunoglobulin G-binding domains of protein G: comparison with calculated values for nuclear magnetic resonance and X-ray structures. Biochemistry, 36:3580–3589. 1997.
87.
KolmRH, SrogaGE, MannervikB. Participation of the phenolic hydroxyl group of Tyr-8 in the catalytic mechanism of human glutathione transferase P1-1. Biochem J, 285,Pt 2:537–540. 1992.
88.
KongstedJ, RydeU, WydraJ, JensenJH. Prediction and rationalization of the pH dependence of the activity and stability of family 11 xylanases. Biochemistry, 46:13581–13592. 2007.
89.
KortemmeT, CreightonTE. Ionisation of cysteine residues at the termini of model alpha-helical peptides. Relevance to unusual thiol pKa values in proteins of the thioredoxin family. J Mol Biol, 253:799–812. 1995.
90.
KoumanovA, KarshikoffA, FriisEP, BorchertTV. Conformational averaging in pK calculations: improvement and limitations in prediction of ionization properties of proteins. J Phys Chem B, 105:9339–9344. 2001.
91.
KoumanovA, RuterjansH, KarshikoffA. Continuum electrostatic analysis of irregular ionization and proton allocation in proteins. Proteins, 46:85–96. 2002.
92.
KrauseG, HolmgrenA. Substitution of the conserved tryptophan 31 in Escherichia coli thioredoxin by site-directed mutagenesis and structure-function analysis. J Biol Chem, 266:4056–4066. 1991.
93.
KrautwurstH, BertiM, EncinasMV, FreyPA. Reaction of wild-type C365S, and C458S saccharomyces cerevisiae phosphoenolpyruvate carboxykinases with fluorescent iodoacetamide derivatives. Arch Biochem Biophys, 327:123–130. 1996.
LahN, LahJ, ZegersI, WynsL, MessensJ. Specific potassium binding stabilizes pI258 arsenate reductase from Staphylococcus aureus. J Biol Chem, 278:24673–24679. 2003.
96.
LappiAK, LensinkMF, AlanenHI, SaloKE, LobellM, JufferAH, RuddockLW. A conserved arginine plays a role in the catalytic cycle of the protein disulphide isomerases. J Mol Biol, 335:283–295. 2004.
97.
LedwidgeR, HongB, DotschV, MillerSM. NmerA of Tn501 mercuric ion reductase: structural modulation of the pKa values of the metal binding cysteine thiols. Biochemistry, 49:8988–8998. 2010.
LewinA, CrowA, HodsonCT, HederstedtL, Le BrunNE. Effects of substitutions in the CXXC active-site motif of the extracytoplasmic thioredoxin ResA. Biochem J, 414:81–91. 2008.
100.
LewinA, CrowA, OubrieA, Le BrunNE. Molecular basis for specificity of the extracytoplasmic thioredoxin ResA. J Biol Chem, 281:35467–35477. 2006.
101.
LiH, HansonC, FuchsJA, WoodwardC, ThomasGJ. Determination of the pKa values of active-center cysteines, cysteines-32 and −35, in Escherichia coli thioredoxin by Raman spectroscopy. Biochemistry, 32:5800–5808. 1993.
102.
LiH, RobertsonAD, JensenJH. The determinants of carboxyl pKa values in turkey ovomucoid third domain. Proteins, 55:689–704. 2004.
103.
LiH, RobertsonAD, JensenJH. Very fast empirical prediction and rationalization of protein pKa values. Proteins, 61:704–721. 2005.
104.
LiY, HuY, ZhangX, XuH, LescopE, XiaB, JinC. Conformational fluctuations coupled to the thiol-disulfide transfer between thioredoxin and arsenate reductase in Bacillus subtilis. J Biol Chem, 282:11078–11083. 2007.
105.
LiuS, ZhangP, JiX, JohnsonWW, GillilandGL, ArmstrongRN. Contribution of tyrosine 6 to the catalytic mechanism of isoenzyme 3–3 of glutathione S-transferase. J Biol Chem, 267:4296–4299. 1992.
106.
LoumayeE, Ferrer-SuetaG, AlvarezB, ReesJF, ClippeA, KnoopsB, RadiR, TrujilloM. Kinetic studies of peroxiredoxin 6 from Arenicola marina: rapid oxidation by hydrogen peroxide and peroxynitrite but lack of reduction by hydrogen sulfide. Arch Biochem Biophys, 514:1–7. 2011.
107.
MacedoMG, AnarB, BronnerIF, CannellaM, SquitieriF, BonifatiV, HoogeveenA, HeutinkP, RizzuP. The DJ-1L166P mutant protein associated with early onset Parkinson's disease is unstable and forms higher-order protein complexes. Hum Mol Genet, 12:2807–2816. 2003.
108.
MarchalS, BranlantG. Evidence for the chemical activation of essential cys-302 upon cofactor binding to nonphosphorylating glyceraldehyde 3-phosphate dehydrogenase from Streptococcus mutans. Biochemistry, 38:12950–12958. 1999.
109.
MarinoSM, GladyshevVN. Cysteine function governs its conservation and degeneration and restricts its utilization on protein surfaces. J Mol Biol, 404:902–916. 2010.
110.
MarinoSM, GladyshevVN. Analysis and functional prediction of reactive cysteine residues. J Biol Chem, 287:4419–4425. 2012.
111.
MasonAC, JensenJH. Protein-protein binding is often associated with changes in protonation state. Proteins, 71:81–91. 2008.
112.
MavridouDA, StevensJM, FergusonSJ, RedfieldC. Active-site properties of the oxidized and reduced C-terminal domain of DsbD obtained by NMR spectroscopy. J Mol Biol, 370:643–658. 2007.
113.
MavridouDA, StevensJM, GoddardAD, WillisAC, FergusonSJ, RedfieldC. Control of periplasmic interdomain thiol:disulfide exchange in the transmembrane oxidoreductase DsbD. J Biol Chem, 284:3219–3226. 2009.
114.
MessensJ, MartinsJC, Van BelleK, BrosensE, DesmyterA, De GieterM, WieruszeskiJM, WillemR, WynsL, ZegersI. All intermediates of the arsenate reductase mechanism, including an intramolecular dynamic disulfide cascade. Proc Natl Acad Sci U S A, 99:8506–8511. 2002.
115.
MessensJ, Van MolleI, VanhaesebrouckP, LimbourgM, Van BelleK, WahniK, MartinsJC, LorisR, WynsL. How thioredoxin can reduce a buried disulphide bond. J Mol Biol, 339:527–537. 2004.
116.
MieyalJJ, StarkeDW, GravinaSA, HocevarBA. Thioltransferase in human red blood cells: kinetics and equilibrium. Biochemistry, 30:8883–8891. 1991.
117.
MossnerE, Huber-WunderlichM, GlockshuberR. Characterization of Escherichia coli thioredoxin variants mimicking the active-sites of other thiol/disulfide oxidoreductases. Protein Sci, 7:1233–1244. 1998.
118.
MossnerE, IwaiH, GlockshuberR. Influence of the pK(a) value of the buried, active-site cysteine on the redox properties of thioredoxin-like oxidoreductases. FEBS Lett, 477:21–26. 2000.
119.
MoutevelisE, WarwickerJ. Prediction of pKa and redox properties in the thioredoxin superfamily. Protein Sci, 13:2744–2752. 2004.
120.
NaorMM, JensenJH. Determinants of cysteine pKa values in creatine kinase and alpha1-antitrypsin. Proteins, 57:799–803. 2004.
121.
NelsonJW, CreightonTE. Reactivity and ionization of the active site cysteine residues of DsbA, a protein required for disulfide bond formation in vivo. Biochemistry, 33:5974–5983. 1994.
122.
NelsonKJ, DayAE, ZengBB, KingSB, PooleLB. Isotope-coded, iodoacetamide-based reagent to determine individual cysteine pK(a) values by matrix-assisted laser desorption/ionization time-of-flight mass spectrometry. Anal Biochem, 375:187–195. 2008.
NordstrandK, ÅslundF, MeunierS, HolmgrenA, OttingG, BerndtKD. Direct NMR observation of the Cys-14 thiol proton of reduced Escherichia coli Glutaredoxin-3 supports the presence of an active site thiol-thiolate hydrogen bond. FEBS letters, 449:196–200. 1999.
130.
OlssonHMM, SøndergaardCR, RostkowskiM, JensenJH. PROPKA3: Consistent treatment of internal and surface residues in empirical pKa predictions. J Chem Theory Comput, 7:525–537. 2011.
131.
PolgarL. Mercaptide-imidazolium ion-pair: the reactive nucleophile in papain catalysis. FEBS Lett, 47:15–18. 1974.
132.
PoratA, LilligCH, JohanssonC, FernandesAP, NilssonL, HolmgrenA, BeckwithJ. The reducing activity of glutaredoxin 3 toward cytoplasmic substrate proteins is restricted by methionine 43. Biochemistry, 46:3366–3377. 2007.
133.
PorterMA, HallJR, LockeJC, JensenJH, MolinaPA. Hydrogen bonding is the prime determinant of carboxyl pKa values at the N-termini of alpha-helices. Proteins, 63:621–635. 2006.
134.
PowersN, JensenJH. Chemically accurate protein structures: Validation of protein NMR structures by comparison of measured and predicted pKa values. J Biomol NMR, 35:39–51. 2006.
135.
PresnellSR, CohenFE. Topological distribution of four-alpha-helix bundles. Proc Natl Acad Sci U S A, 86:6592–6596. 1989.
136.
QinJ, CloreGM, GronenbornAM. The high-resolution three-dimensional solution structures of the oxidized and reduced states of human thioredoxin. Structure, 2:503–522. 1994.
137.
RablenPR, LockmanJW, JorgensenWL. Ab initio study of hydrogen-bonded complexes of small organic molecules with water. J Phys Chem A, 102:3782–3797. 1998.
138.
ReckenfelderbaumerN, Krauth-SiegelRL. Catalytic properties, thiol pK value, and redox potential of Trypanosoma brucei tryparedoxin. J Biol Chem, 277:17548–17555. 2002.
139.
RenG, StephanD, XuZ, ZhengY, TangD, HarrisonRS, KurzM, JarrottR, ShouldiceSR, HinikerA, MartinJL, HerasB, BardwellJC. Properties of the thioredoxin fold superfamily are modulated by a single amino acid residue. J Biol Chem, 284:10150–10159. 2009.
140.
RoosG, ButsL, Van BelleK, BrosensE, GeerlingsP, LorisR, WynsL, MessensJ. Interplay between ion binding and catalysis in the thioredoxin-coupled arsenate reductase family. J Mol Biol, 360:826–838. 2006.
141.
RoosG, FoloppeN, Van LaerK, WynsL, NilssonL, GeerlingsP, MessensJ. How thioredoxin dissociates Its mixed disulfide. PloS Comp Biol, 5:e1000461. 2009.
142.
RoosG, Garcia-PinoA, Van BelleK, BrosensE, WahniK, VandenbusscheG, WynsL, LorisR, MessensJ. The conserved active site proline determines the reducing power of Staphylococcus aureus thioredoxin. J Mol Biol, 368:800–811. 2007.
143.
RoosG, LoverixS, GeerlingsP. Origin of the pK(a) perturbation of N-terminal cysteine in alpha- and 3(10)-helices: a computational DFT study. J Phys Chem B, 110:557–562. 2006.
144.
RoosG, MessensJ. Protein sulfenic acid formation: from cellular damage to redox regulation. Free Radic Biol Med, 51:314–326. 2011.
145.
RoosG, MessensJ, LoverixS, WynsL, GeerlingsP. A computational and conceptual DFT study on the Michaelis complex of pI258 arsenate reductase. Structural aspects and activation of the electrophile and nucleophile. J Phys Chem B, 108:17216–17225. 2004.
146.
RuddockLW, HirstTR, FreedmanRB. pH-dependence of the dithiol-oxidizing activity of DsbA (a periplasmic protein thiol:disulphide oxidoreductase) and protein disulphide-isomerase: studies with a novel simple peptide substrate. Biochem J, 315,Pt 3:1001–1005. 1996.
147.
RullmannJA, BellidoMN, van DuijnenPT. The active site of papain. All-atom study of interactions with protein matrix and solvent. J Mol Biol, 206:101–118. 1989.
SanchezR, RiddleM, WooJ, MomandJ. Prediction of reversibly oxidized protein cysteine thiols using protein structure properties. Protein Sci, 17:473–481. 2008.
150.
SanchoJ, SerranoL, FershtAR. Histidine residues at the N- and C-termini of alpha-helices: perturbed pKas and protein stability. Biochemistry, 31:2253–2258. 1992.
151.
SchlesingerP, WestleyJ. An expanded mechanism for rhodanese catalysis. J Biol Chem, 249:780–788. 1974.
ShekhterT, MetanisN, DawsonPE, KeinanE. A residue outside the active site CXXC motif regulates the catalytic efficiency of Glutaredoxin 3. Mol BioSyst, 6:241–248. 2010.
156.
SheridanRP, LevyRM, SalemmeFR. alpha-Helix dipole model and electrostatic stabilization of 4-alpha-helical proteins. Proc Natl Acad Sci U S A, 79:4545–4549. 1982.
157.
SimonsonT, CarlssonJ, CaseDA. Proton binding to proteins: pK(a) calculations with explicit and implicit solvent models. J Am Chem Soc, 126:4167–4180. 2004.
158.
SimonsonT, PerahiaD. Dielectric properties of proteins from simulations: tools and techniques. Comput Phys Commun, 91:291–303. 1995.
159.
SimonsonT, PerahiaD. Internal and interfacial dielectric properties of cytochrome c from molecular dynamics in aqueous solution. Proc Natl Sci U S A, 92:1082–1086. 1995.
160.
SøndergaardCR, OlssonHMM, RostkowskiM, JensenJH. Improved treatment of ligands and coupling effects in empirical calculation and rationalization of pKa values. J Chem Theory Comput, 7:2284–2295. 2011.
161.
StirnimannCU, RozhkovaA, GrauschopfU, BockmannRA, GlockshuberR, CapitaniG, GrutterMG. High-resolution structures of Escherichia coli cDsbD in different redox states: a combined crystallographic, biochemical and computational study. J Mol Biol, 358:829–845. 2006.
162.
SubramanianS, RossPD. The enthalpy of protolysis of liver alcohol dehydrogenase upon binding nicotinamide adenine dinucleotide. J Biol Chem, 254:7826–7830. 1979.
163.
SunXX, WangCC. The N-terminal sequence (residues 1–65) is essential for dimerization, activities, and peptide binding of Escherichia coli DsbC. J Biol Chem, 275:22743–22749. 2000.
164.
SzajewskiRP, WhitesidesGM. Rate constants and equilibrium constants for thiol-disulfide interchange reactions involving oxidized glutathione. J Am Chem Soc, 102:2011–2025. 1980.
165.
TajcSG, TolbertBS, BasavappaR, MillerBL. Direct determination of thiol pKa by isothermal titration microcalorimetry. J Am Chem Soc, 126:10508–10509. 2004.
166.
TakahashiN, CreightonTE. On the reactivity and ionization of the active site cysteine residues of Escherichia coli thioredoxin. Biochemistry, 35:8342–8353. 1996.
167.
TaylorR, KennardO. Crystallographic evidence for the existence of C-H…O, C-H…N, and C-H…Cl hydrogen bonds. J Am Chem Soc, 104:5063–5070. 1982.
168.
ThomasKA, SmithGM, ThomasTB, FeldmannRJ. Electronic distributions within protein phenylalanine aromatic rings are reflected by the three-dimensional oxygen atom environments. Proc Natl Acad Sci U S A, 79:4843–4847. 1982.
169.
ThurlkillRL, GrimsleyGR, ScholtzJM, PaceCN. pK values of the ionizable groups of proteins. Protein Sci, 15:1214–1218. 2006.
170.
UllmannGM, NoodlemanL, CaseDA. Density functional calculation of p K(a) values and redox potentials in the bovine Rieske iron-sulfur protein. J Biol Inorg Chem, 7:632–639. 2002.
171.
van DuijnenPT, TholeBT, HolWG. On the role of the active site helix in papain, an ab initio molecular orbital study. Biophys Chem, 9:273–280. 1979.
172.
van StraatenM, MissiakasD, RainaS, DarbyNJ. The functional properties of DsbG, a thiol-disulfide oxidoreductase from the periplasm of Escherichia coli. FEBS Lett, 428:255–258. 1998.
173.
van VlijmenHW, SchaeferM, KarplusM. Improving the accuracy of protein pKa calculations: conformational averaging versus the average structure. Proteins, 33:145–158. 1998.
174.
VargasR, GarzaD, DixonA, HayBP. How strong is the Ca-H…O=C hydrogen bond?J Am Chem Soc, 122:4750–4755. 2000.
175.
VilladangosAF, Van BelleK, WahniK, Tamu DufeV, FreitasS, NurH, De GalanS, GilJA, ColletJF, MateosLM, MessensJ. Corynebacterium glutamicum survives arsenic stress with arsenate reductases coupled to two distinct redox mechanisms. Mol Microbiol, 82:998–1014. 2011.
176.
WadaA. The alpha-helix as an electric macro-dipole. Adv Biophys, 1–63. 1976.
177.
WangPF, McLeishMJ, KneenMM, LeeG, KenyonGL. An unusually low pK(a) for Cys282 in the active site of human muscle creatine kinase. Biochemistry, 40:11698–11705. 2001.
178.
WangRW, NewtonDJ, HuskeySE, McKeeverBM, PickettCB, LuAY. Site-directed mutagenesis of glutathione S-transferase YaYa. Important roles of tyrosine 9 and aspartic acid 101 in catalysis. J Biol Chem, 267:19866–19871. 1992.
179.
WarwickerJ, GanePJ. Calculation of Cys 30 ΔpKa's and oxidising power for DsbA mutants. FEBS Lett, 385:105–108. 1996.
180.
WarwickerJ, WatsonHC. Calculation of the electric potential in the active site cleft due to alpha-helix dipoles. J Mol Biol, 157:671–679. 1982.
181.
WatersML. Aromatic interactions in model systems. Curr Opin Chem Biol, 6:736–741. 2002.
182.
WennmohsF, StaemmlerV, SchindlerM. Theoretical investigation of weak hydrogen bonds to sulfur. J Chem Phys, 119:3208–3218. 2003.
183.
WinterbournCC, MetodiewaD. Reactivity of biologically important thiol compounds with superoxide and hydrogen peroxide. Free Radic Biol Med, 27:322–328. 1999.
184.
WittAC, LakshminarasimhanM, RemingtonBC, HasimS, PozharskiE, WilsonMA. Cysteine pKa depression by a protonated glutamic acid in human DJ-1. Biochemistry, 47:7430–7440. 2008.
185.
WlodekST, AntosiewiczJ, McCammonJA. Prediction of titration properties of structures of a protein derived from molecular dynamics trajectories. Protein Sci, 6:373–382. 1997.
186.
WooHA, JeongW, ChangTS, ParkKJ, ParkSJ, YangJS, RheeSG. Reduction of cysteine sulfinic acid by sulfiredoxin is specific to 2-cys peroxiredoxins. J Biol Chem, 280:3125–3128. 2005.
187.
XiaoG, LiuS, JiX, JohnsonWW, ChenJ, ParsonsJF, StevensWJ, GillilandGL, ArmstrongRN. First-sphere and second-sphere electrostatic effects in the active site of a class mu gluthathione transferase. Biochemistry, 35:4753–4765. 1996.
188.
YangA-S, GunnerMR, SampognaR, SharpK, HonigB. On the Calculation of pKas in Proteins. Proteins, 15:252–265. 1993.
189.
YangAS, HonigB. On the pH dependence of protein stability. J Mol Biol, 231:459–474. 1993.
190.
YangYF, WellsWW. Identification and characterization of the functional amino acids at the active center of pig liver thioltransferase by site-directed mutagenesis. J Biol Chem, 266:12759–12765. 1991.
191.
YeJ, ChoSH, FuselierJ, LiW, BeckwithJ, RapoportTA. Crystal structure of an unusual thioredoxin protein with a zinc finger domain. J Biol Chem, 282:34945–34951. 2007.
192.
YouTJ, BashfordD. Conformation and hydrogen ion titration of proteins: a continuum electrostatic model with conformational flexibility. Biophys J, 69:1721–1733. 1995.
193.
ZapunA, MissiakasD, RainaS, CreightonTE. Structural and functional characterization of DsbC, a protein involved in disulfide bond formation in Escherichia coli. Biochemistry, 34:5075–5089. 1995.
194.
ZegersI, MartinsJC, WillemR, WynsL, MessensJ. Arsenate reductase from S. aureus plasmid pI258 is a phosphatase drafted for redox duty. Nat Struct Biol, 8:843–847. 2001.
195.
ZhengF, ZhanC-G, OrnsteinRL. Theoretical determination of two structural forms of the active site in cadmium-containing phosphotriesterases. J Phys Chem B, 106:717–722. 2002.
196.
ZhouP, TianF, LvF, ShangZ. Geometric characteristics of hydrogen bonds involving sulfur atoms in proteins. Proteins, 76:151–163. 2009.