The basics of thiols and cysteines in redox biology and chemistry

Abstract

Cysteine is one of the least abundant amino acids, yet it is frequently found as a highly conserved residue within functional (regulatory, catalytic, or binding) sites in proteins. It is the unique chemistry of the thiol or thiolate group of cysteine that imparts to functional sites their specialized properties (e.g., nucleophilicity, high-affinity metal binding, and/or ability to form disulfide bonds). Highlighted in this review are some of the basic biophysical and biochemical properties of cysteine groups and the equations that apply to them, particularly with respect to pKa and redox potential. Also summarized are the types of low-molecular-weight thiols present in high concentrations in most cells, as well as the ways in which modifications of cysteinyl residues can impart or regulate molecular functions important to cellular processes, including signal transduction.

Keywords: Cysteine; Free radicals; Redox potential; Redox regulation; Thiols; pK(a).

Figure 1

Structures of cysteinyl and selenocysteinyl residues within proteins. The aminoacyl groups are shown to the left, with dotted lines representing peptide bonds to the next residue on either side. Both protonated (left) and deprotonated (right) forms of these amino acids are depicted with average pK_a values (that can vary in particular protein microenvironments).

Figure 2

Biologically relevant thiol-containing small molecules. Red highlights the cysteinyl moiety in glutathione and the spermidine linker in trypanothione. Not shown is glutathione amide (found in some γ-proteobacteria), which includes an amide rather than carboxylate group on the Gly of glutathione.

Figure 3

Cysteine is the least exposed residue in proteins, yet its chemical–physical properties are those of a polar residue. (a) Cys exposure was calculated for approximately 15,000 nonredundant proteins from PDB as described in Marino & Gladyshev, 2010. The percentage of burial is shown for each composing atom of Cys and, for comparison, for each atom in Ser, Ala, Thr, and Met. Above each point, the positions along the side chain are reported (i.e., Cα, Cβ, Cγ, Cδ, and Cε). Note that Thr is Cβ branched (i.e., has two γ atoms and no δ atom). (b) Calculations with a QM approach are plotted as a function of atoms composing an amino acid residue. For comparison, residues with charge distributions similar to that of Cys are shown (data provided by Dr. Annick Thomas and Dr. Robert Brasseur). Reprinted with permission from Marino & Gladyshev (2010). Copyright 2010, Elsevier Ltd.

Figure 4

pH titrations to determine thiol pK_a, and population shifts over biological pH ranges relative to thiol pK_a. Absorbance data collected at 240 nm can in some cases be used to track thiolate formation at various pH values (with fits conducted using the equation given in the text). Using the Henderson-Hasselbalch equation (also in the text), ratios of the populations of protonated and deprotonated species can be calculated given the pK_a of the thiol of interest and the pH of the system (shown in table).

Figure 5

Modified and oxidized products of thiol groups. Shown are some of the biologically significant modifications occurring on small molecule and protein thiols, including 1 and 2 electron oxidized forms as well as adducts formed with electrophiles (X). Species in red may not be recoverable biologically; one exception is R-SO₂⁻ in peroxiredoxins which is recycled by a dedicated repair protein, sulfiredoxin. Chemical pathways shown are representative mechanisms and are not meant to imply that these are the only or most important biological reactions. Dotted lines and brackets are used to indicate overall chemical relationships between groups, but not necessarily specific chemical pathways.

Figure 6

Biological oxidants of thiol groups. Although neutrophils and macrophages were the first cell types recognized to generate cellular oxidants as an essential part of the body’s immune defense, it has subsequently been recognized that there are many sources of oxidants present in many other cell types, as well (see text for details).

Figure 7

Electron flow from reduced pyridine nucleotides (NADPH and NADH) and flavin-based reductase systems to reductively recycle key oxidized proteins in cells. Among the reductase systems are the thioredoxin (Trx) system, including thioredoxin reductase (TrxR), the glutathione (GSH) system including glutathione reductase which recycles oxidized glutathione (GSSG), in some cases with the help of glutaredoxins (Grx), and sets of specialized microbial systems with a more restricted distribution in biology. This includes the bacterial peroxiredoxin (AhpC) regenerator AhpF, the clostridial system Cp34 (a Trx reductase homologue) and Cp9 (a Grx homologue), and the trypanothione (TrySH) system, including its reductase (TryR) and the Trx-like small protein tryparedoxin (Txn).