Redox-active Cysteine Residues Research Articles

How Thioredoxin can Reduce a Buried Disulphide Bond

We present a study of the interaction between thioredoxin and the model enzyme pI258 arsenate reductase (ArsC) from Staphylococcus aureus. ArsC catalyses the reduction of arsenate to arsenite. Three redox active cysteine residues (Cys10, Cys82 and Cys89) are involved. After a single catalytic arsenate reduction event, oxidized ArsC exposes a disulphide bridge between Cys82 and Cys89 on a looped-out redox helix. Thioredoxin converts oxidized ArsC back towards its initial reduced state. In the absence of a reducing environment, the active-site P-loop of ArsC is blocked by the formation of a second disulphide bridge (Cys10–Cys15). While fully reduced ArsC can be recovered by exposing this double oxidized ArsC to thioredoxin, the P-loop disulphide bridge is itself inaccessible to thioredoxin. To reduce this buried Cys10–Cys15 disulphide-bridge in double oxidized ArsC, an intra-molecular Cys10–Cys82 disulphide switch connects the thioredoxin mediated inter-protein thiol–disulphide transfer to the buried disulphide. In the initial step of the reduction mechanism, thioredoxin appears to be selective for oxidized ArsC that requires the redox helix to be looped out for its interaction. The formation of a buried disulphide bridge in the active-site might function as protection against irreversible oxidation of the nucleophilic cysteine, a characteristic that has also been observed in the structurally similar low molecular weight tyrosine phosphatase.

Structure of the Large Subunit of Class Ib Ribonucleotide Reductase from Salmonella typhimurium and its Complexes with Allosteric Effectors

The three-dimensional structure of the large subunit of the first member of a class Ib ribonucleotide reductase, R1E of Salmonella typhimurium, has been determined in its native form and together with three allosteric effectors. The enzyme contains the characteristic ten-stranded α/β-barrel with catalytic residues at a finger loop in its center and with redox-active cysteine residues at two adjacent barrel strands. Structures where the redox-active cysteine residues are in reduced thiol form and in oxidized disulfide form have been determined revealing local structural changes. The R1E enzyme differs from the class Ia enzyme, Escherichia coli R1, by not having an overall allosteric regulation. This is explained from the structure by differences in the N-terminal domain, which is about 50 residues shorter and lacks the overall allosteric binding site. R1E has an allosteric substrate specificity regulation site and the binding site for the nucleotide effectors is located at the dimer interface similarly as for the class Ia enzymes. We have determined the structures of R1E in the absence of effectors and with dTTP, dATP and dCTP bound. The low affinity for ATP at the specificity site is explained by a tyrosine, which hinders nucleotides containing a 2′-OH group to bind.

C-propeptide region of human pro α 1 type 1 collagen interacts with thioredoxin

Thioredoxin (TRX) is one of major components of thiol reducing systems. To investigate the molecular mechanism of TRX function in the lung tissue, we screened a human lung epithelial cell cDNA library for TRX-binding protein by yeast two-hybrid systems. We isolated a plasmid containing C-propeptide region of human pro α 1 type 1 collagen (CP-pro α 1(1)). CP-pro α 1(1) stably binds to wild type TRX but not to mutant TRX, in which redox-active cysteine residues are substituted. Failure of the interaction of mutant TRX with CP-pro α 1(1) was confirmed in yeast two-hybrid systems. The CP-pro α 1(1)/TRX interaction was increased by dithiothreitol treatment, but was markedly inhibited by hydrogen peroxide or diamide treatment. These data showed that the reducing status of TRX active site cysteine residues is important for the TRX–CP-pro α 1(1) interaction, indicating that collagen biosynthesis is under the regulation of TRX-dependent redox control.

Molecular Characterization of the Arabidopsis thaliana Flavoprotein AtHAL3a Reveals the General Reaction Mechanism of 4′-Phosphopantothenoylcysteine Decarboxylases

The Arabidopsis thaliana flavoprotein AtHAL3a, which is linked to plant growth and salt and osmotic tolerance, catalyzes the decarboxylation of 4'-phosphopantothenoylcysteine to 4'-phosphopantetheine, a key step in coenzyme A biosynthesis. AtHAL3a is similar in sequence and structure to the LanD enzymes EpiD and MrsD, which catalyze the oxidative decarboxylation of peptidylcysteines. Therefore, we hypothesized that the decarboxylation of 4'-phosphopantothenoylcysteine also occurs via an oxidatively decarboxylated intermediate containing an aminoenethiol group. A set of AtHAL3a mutants were analyzed to detect such an intermediate. By exchanging Lys(34), we found that AtHAL3a is not only able to decarboxylate 4'-phosphopantothenoylcysteine but also pantothenoylcysteine to pantothenoylcysteamine. Exchanging residues within the substrate binding clamp of AtHAL3a (for example of Gly(179)) enabled the detection of the proposed aminoenethiol intermediate when pantothenoylcysteine was used as substrate. This intermediate was characterized by its high absorbance at 260 and 280 nm, and the removal of two hydrogen atoms and one molecule of CO(2) was confirmed by ultrahigh resolution mass spectrometry. Using the mutant AtHAL3a C175S enzyme, the product pantothenoylcysteamine was not detectable; however, oxidatively decarboxylated pantothenoylcysteine could be identified. This result indicates that reduction of the aminoenethiol intermediate depends on a redox-active cysteine residue in AtHAL3a.

Specific and Reversible Inactivation of Phycomyces blakesleeanus Isocitrate Lyase by Ascorbate-Iron: Role of Two Redox-Active Cysteines

Phycomyces blakesleeanus isocitrate lyase (EC 4.1.3.1) is in vivo reversibly inactivated by hydrogen peroxide. The purified enzyme showed reversible inactivation by an ascorbate plus Fe2+ system under aerobic conditions. Inactivation requires hydrogen peroxide; was prevented by catalase, EDTA, Mg2+, isocitrate, GSH, DTT, or cysteine; and was reversed by thiols. The ascorbate served as a source of hydrogen peroxide and also reduced the Fe3+ ions produced in a “site-specific” Fenton reaction. Two redox-active cysteine residues per enzyme subunit are targets of oxidative modification; one of them is located at the catalytic site and the other at the metal regulatory site. The oxidized enzyme showed covalent and conformational changes that led to inactivation, decreased thermal stability, and also increased inactivation by trypsin. These results represent an example of redox regulation of an enzymatic activity, which may play a role as a sensor of redox cellular status.

Identification of Thioredoxin-binding Protein-2/Vitamin D3 Up-regulated Protein 1 as a Negative Regulator of Thioredoxin Function and Expression

Recent works have shown the importance of reduction/oxidation (redox) regulation in various biological phenomena. Thioredoxin (TRX) is one of the major components of the thiol reducing system and plays multiple roles in cellular processes such as proliferation, apoptosis, and gene expression. To investigate the molecular mechanism of TRX action, we used a yeast two-hybrid system to identify TRX-binding proteins. One of the candidates, designated as thioredoxin-binding protein-2 (TBP-2), was identical to vitamin D(3) up-regulated protein 1 (VDUP1). The association of TRX with TBP-2/VDUP1 was observed in vitro and in vivo. TBP-2/VDUP1 bound to reduced TRX but not to oxidized TRX nor to mutant TRX, in which two redox active cysteine residues are substituted by serine. Thus, the catalytic center of TRX seems to be important for the interaction. Insulin reducing activity of TRX was inhibited by the addition of recombinant TBP-2/VDUP1 protein in vitro. In COS-7 and HEK293 cells transiently transfected with TBP-2/VDUP1 expression vector, decrease of insulin reducing activity of TRX and diminishment of TRX expression was observed. These results suggested that TBP-2/VDUP1 serves as a negative regulator of the biological function and expression of TRX. Treatment of HL-60 cells with 1alpha, 25-dihydroxyvitamin D(3) caused an increase of TBP-2/VDUP1 expression and down-regulation of the expression and the reducing activity of TRX. Therefore, the TRX-TBP-2/VDUP1 interaction may be an important redox regulatory mechanism in cellular processes, including differentiation of myeloid and macrophage lineages.

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

The active site of Escherichia coli glutaredoxin-3 (Grx3) consists of two redox active cysteine residues in the sequence -C 11-P-Y-C 14-H-. The 1H NMR resonance of the cysteine thiol proton of Cys-14 in reduced Grx3 is observed at 7.6 ppm. The large downfield shift and NOEs observed with this thiol proton resonance suggest the presence of a hydrogen bond with the Cys-11 thiolate, which is shown to have an abnormally low p K a value. A hydrogen bond would also agree with activity data of Grx3 active site mutants. Furthermore, the activity is reduced in a Grx3 H15V mutant, indicating electrostatic contributions to the stabilization of the Cys-11 thiolate.

Tumor Promoter Benzoyl Peroxide Induces Sulfhydryl Oxidation in Protein Kinase C: Its Reversibility Is Related to the Cellular Resistance to Peroxide-Induced Cytotoxicity

Since tumor promoter benzoyl peroxide (BPO) mimics phorbol esters in some aspects, its effects on protein kinase C (PKC) were previously studied. However, in those studies due to the presence of thiol agents in the PKC preparations, the sensitive reaction of BPO with redox-active cysteine residues in PKC was not observed. In this study, by excluding thiol agents present in the purified PKC preparation, low concentrations of BPO modified PKC, resulting in the loss of both kinase activity and phorbol ester binding (IC50= 0.2 to 0.5 μM). This modification, which was not dependent on transition metals, was totally blocked by a variety of thiol agents including GSH, which directly reacted with BPO. Substoichiometric amounts of BPO (0.4 mol/mol of PKC) oxidized two sulfhydryls in PKC and inactivated the enzyme which was readily reversed by dithiothreitol. The regulatory domain having zinc thiolate structures supporting the membrane-inserting region provided the specificity for PKC reaction with BPO, which partitioned into the membrane. Unlike H2O2, BPO did not induce the generation of the Ca2+/lipid-independent activated form of PKC. Other redox-sensitive enzymes such as protein kinase A, phosphorylase kinase, and protein phosphatase 2A required nearly 25- to 100-fold higher concentrations of BPO for inactivation. BPO also inactivated PKC in a variety of cell types. In the JB6 (30 P−) nonpromotable cell line and other normal cell lines, where BPO was more cytotoxic, it readily inactivated PKC due to a slow reversibility of this inactivation by the cell. However, in the JB6 (41 P+) promotable cell line, C3H10T1/2 and B16 melanoma cells, where BPO was less cytotoxic, it did not readily inactivate PKC due to a rapid reversibility of this inactivation by an endogenous mechanism. Nevertheless, BPO inactivated PKC at an equal rate in the homogenates prepared from all these cell types. Inclusion of NADPH reversed this inactivation in the homogenates to a different extent, presumably due to a difference in distribution of a protein disulfide reductase, which reverses this oxidative modification. BPO-induced modification of PKC occurred independent of the cellular status of GSH. However, externally added GSH and cell-impermeable thiol agents prevented the BPO-induced modification of PKC. Since BPO readily partitions into membranes, its reaction with redox-cycling thiols of membrane proteins such as PKC may trigger epigenetic events to prevent cytotoxicity, but favor tumor promotion.

Assignment of 1H, 13C, and 15N resonances of reduced Escherichia coli glutaredoxin 2.

The thioredoxin and glutaredoxin families participate as hydrogen donors in a wide variety of cellular reactions (Holmgren, 1989). Glutaredoxins are distinguished from thioredoxins by their ability to catalyze glutathione (GSH)-disulfide oxidoreductions via two redox-active cysteine residues (Holmgren and Aslund, 1995). Glutaredoxin 2 (Grx2) is one of the three glutaredoxins found in E. coli, but unlike the other two, Grx2 is not a hydrogen donor for ribonucleotide reductase (Aslund et al., 1994). It has an unusually large size (24.3 kDa) with 215 amino acid residues, whereas the typical molecular weights for glutaredoxins are around 10 kDa. Grx2 has little sequence homology with other glutaredoxins or thioredoxins, except it contains the conserved active site sequence of Cys-Pro-Tyr-Cys. Grx2 appears most abundant among all three glutaredoxins in E. coli. It may account for about 80% of the glutaredoxin activity in the GSH-disulfide oxidoreductase assay (Vlamis-Gardikas et al., 1997). Although the specific biological function is still unknown, the unique properties of Grx2 indicate that it is likely to have a distinctive function. Grx2 has been overproduced in E. coli; the protein was doubly labeled with 13C and 15N, and triply labeled with 2H, 13C and 15N. Multidimensional NMR spectroscopic studies of the labeled Grx2 have led to extensive backbone and side-chain resonance assignments.

Cancer-Preventive Selenocompounds Induce a Specific Redox Modification of Cysteine-Rich Regions in Ca2+- Dependent Isoenzymes of Protein Kinase C

Since protein kinase C (PKC) serves as a receptor for phorbol ester type tumor promoters and oxidants and has unique redox-active cysteine-rich regions, we have determined whether various chemopreventive selenocompounds could affect this enzyme. At lower concentrations, selenite decreased the kinase activity (IC50= 0.5 μm), while at higher concentrations it decreased phorbol ester binding. However, when the catalytic and regulatory domains of PKC were separated by proteolysis, the catalytic domain retained its sensitivity to selenite, while the regulatory domain lost its sensitivity. Cysteine residues were quantitated in PKC modified with selenite by using 5,5′-dithiobis(2-nitrobenzoic acid) and also by using 2-nitro-5-thiosulfobenzoic acid after sulfitolysis. At lower concentrations, selenite induced a modification of four cysteine residues resulting in the formation of two disulfides, while at higher concentrations it induced a modification of seven to eight cysteine residues resulting in the formation of three to four disulfides. Contrary to selenite, selenocystine and selenodiglutathione (GSSeSG) readily inactivated the kinase activity, but not the phorbol ester binding. These two agents induced a two-stage modification of PKC; a limited modification at low concentrations leads to a loss of affinity for ATP, while an excessive modification at high concentrations leads to a loss ofVmax. Selenocystine and GSSeSG were 100,000-fold more potent than GSSG in inactivating PKC. The isoenzymes α, β, and γ exhibited an identical susceptibility to these selenocompounds. These results suggested that the cysteine residues present within the catalytic domain of these isoenzymes, although apart in the sequence, may be clustered in the tertiary structure to react with selenite, as well as may be in close proximity to some of the cysteines in the regulatory domain. Selenite did not affect protein kinase A, whereas GSSeSG and selenocystine inactivated the catalytic subunit after dissociation from the regulatory subunit at concentrations 100- and 800-fold, respectively, higher than that required for PKC inactivation. All three selenocompounds did not affect the activities of phosphorylase kinase and protein phosphatase 2A. Taken together, these results suggest that the accessible redox-active cysteine residues present in the PKC catalytic domain can react with certain specificity with redox-active selenocompounds such as selenite, selenocystine, and GSSeSG relative to other protein kinases tested.

Binding of allosteric effectors to ribonucleotide reductase protein R1: reduction of active-site cysteines promotes substrate binding.

Ribonucleotide reductase (RNR) is an essential enzyme in DNA synthesis, catalyzing all de novo synthesis of deoxyribonucleotides. The enzyme comprises two dimers, termed R1 and R2, and contains the redox active cysteine residues, Cys462 and Cys225. The reduction of ribonucleotides to deoxyribonucleotides involves the transfer of free radicals. The pathway for the radical has previously been suggested from crystallographic results, and is supported by site-directed mutagenesis studies. Most RNRs are allosterically regulated through two different nucleotide-binding sites: one site controls general activity and the other controls substrate specificity. Our aim has been to crystallographically demonstrate substrate binding and to locate the two effector-binding sites. We report here the first crystal structure of RNR R1 in a reduced form. The structure shows that upon reduction of the redox active cysteines, the sulfur atom of Cys462 becomes deeply buried. The more accessible Cys225 moves to the former position of Cys462 making room for the substrate. In addition, the structures of R1 in complexes with effector, effector analog and effector plus substrate provide information about these binding sites. The substrate GDP binds in a cleft between two domains with its beta-phosphate bound to the N termini of two helices; the ribose forms hydrogen bonds to conserved residues. Binding of dTTP at the allosteric substrate specificity site stabilizes three loops close to the dimer interface and the active site, whereas the general allosteric binding site is positioned far from the active site. Binding of substrate at the active site of the enzyme is structurally regulated in two ways: binding of the correct substrate is regulated by the binding of allosteric effectors and binding of the actual substrate occurs primarily when the active-site cysteines are reduced. One of the loops stabilized upon binding of dTTP participates in the formation of the substrate-binding site through direct interaction with the nucleotide base. The general allosteric effector site, located far from the active site, appears to regulate subunit interactions within the holoenzyme.

Homology Modelling of a Newly Discovered Thioredoxin Protein and Analysis of the Force Field and Electrostatic Properties

Thioredoxin is a small protein (Mr approximately 12,000) found in all living cells from archaebacteria to humans. The active site is highly conserved and has two redox-active cysteine residues in the sequence: -Trp-Cys-Gly-Pro-Cys-. Besides the function of the reduced form as a powerful protein disulfide oxidoreductase, thioredoxin is known to regulate and activate different target enzymes, i.e. ribonucleotide reductase and the mitochondrial 2-oxoacid dehydrogenase multienzyme complexes. Despite the high degree of homology between thioredoxin proteins from different species, there exists a strong variation in the capability of activating target enzymes. This is yet unexplainable, since there still exists no model of a thioredoxin/receptor complex.

Human IgG is substrate for the thioredoxin system: Differential cleavage pattern of interchain disulfide bridges in IgG subclasses

Thioredoxin, a 12,000 mol. wt protein with two redox-active cysteine residues, together with thioredoxin reductase and NADPH, may reduce protein disulfides and thereby act as a molecular probe of their structure and reactivity. Interchain and intrachain disulfides are structural elements in all immunoglobulins and therefore potential substrates for the reduced thioredoxin, Trx(SH) 2. It was investigated whether such disulfides are cleaved in human polyclonal IgG and IgG subclass myeloma proteins by both the human and the Escherishia coli thioredoxin systems. The reactions were monitored spectrophotometrically as oxidation of NADPH at 340 nm, and by following the kinetics of the cleavage patterns with sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE). Human IgG was a substrate for both prokaryotic and eukaryotic Trx(SH) 2, which directly reduced IgG disulfides in a time and dose-dependent manner. Stoichiometric analyses indicated near-complete reduction of mainly inter-heavy-light chain and inter-heavy chain disulfides, and SDS-PAGE corroborated that the buried intrachain disulfides were left intact. The kinetic studies showed that IgG1, IgG3 and IgG4 were readily reduced into heavy and light chains via the formation of half-molecules with slightly slower kinetics for IgG4. In sharp contrast. IgG2 was not cleaved at all, even with increased thioredoxin concentrations or reduction times. A small but significant NADPH consumption by IgG2 myeloma proteins suggested reduction of a labile interchain or surface-exposed mixed disulfide. Consistent results were obtained for several IgG myeloma proteins within each subclass. The structural and functional importance of interchain disulfides in immunoglobulins suggests physiological implications of the thioredoxin system.

Cloning, Overexpression, and Characterization of Glutaredoxin 2, An Atypical Glutaredoxin from Escherichia coli

Glutaredoxin 2 (Grx2) from Escherichia coli catalyzes GSH-disulfide oxidoreductions via two redox-active cysteine residues, but in contrast to glutaredoxin 1 (Grx1) and glutaredoxin 3 (Grx3), is not a hydrogen donor for ribonucleotide reductase. To characterize Grx2, a chromosomal fragment containing the E. coli Grx2 gene (grxB) was cloned and sequenced. grxB (645 base pairs) is located between the rimJ and pyrC genes while an open reading frame immediately upstream grxB encodes a novel transmembrane protein of 402 amino acids potentially belonging to class II of substrate export transporters. The deduced amino acid sequence for Grx2 comprises 215 residues with a molecular mass of 24.3 kDa. There is almost no similarity between the amino acid sequence of Grx2 and Grx1 or Grx3 (both 9-kDa proteins) with the exception of the active site which is identical in all three glutaredoxins (C9PYC12 for Grx2). Only limited similarities were noted to glutathione S-transferases (Grx2 amino acids 16-72), and protein disulfide isomerases from different organisms (Grx2 amino acids 70-180). Grx2 was overexpressed and purified to homogeneity and its activity was compared with those of Grx1 and Grx3 using GSH, NADPH, and glutathione reductase in the reduction of 0.7 mM beta-hydroxyethyl disulfide. The three glutaredoxins had similar apparent Km values for GSH (2-3 mM) but Grx2 had the highest apparent kcat (554 s-1). Expression of two truncated forms of Grx2 (1-114 and 1-133) which have predicted secondary structures similar to Grx1 (betaalphabetaalphabetabetaalpha) gave rise to inclusion bodies. The mutant proteins were resolubilized and purified but lacked GSH-disulfide oxidoreductase activity. The latter should therefore require the participation of amino acid residues from the COOH-terminal half of the molecule and is probably not confined to a Grx1-like NH2-terminal subdomain. Grx2 being radically different from the presently known glutaredoxins in terms of molecular weight, amino acid sequence, catalytic activity, and lack of a consensus GSH-binding site is the first member of a novel class of glutaredoxins.

Cloning, sequence, and properties of the soluble pyridine nucleotide transhydrogenase of Pseudomonas fluorescens.

The gene encoding the soluble pyridine nucleotide transhydrogenase (STH) of Pseudomonas fluorescens was cloned and expressed in Escherichia coli. STH is related to the flavoprotein disulfide oxidoreductases but lacks one of the conserved redox-active cysteine residues. The gene is highly similar to an E. coli gene of unknown function.

The Ten-stranded β/α Barrel in Ribonucleotide Reductase Protein R1

The large subunit of ribonucleotide reductase (RNR) contains a ten-stranded β/α barrel of a new type consisting of two antiparallel halves. The two halves of the barrel are pseudo 2-fold-related, have similar folds but different additional intervening secondary structure elements and loops. The inner diameter of the RNR barrel, 15 Å to 20 Å, is significantly larger than for the (βα) 8barrels. The larger barrel forms a stable framework which holds an inserted hairpin loop rigidly and exposes active site residues at its tip. The barrel organization allows three cysteine residues to be positioned close to each other without forming unfavorable disulfide bridges between Cys439 on the tip of the inserted loop and the redox-active cysteine residues on the barrel strands. Redox-active cysteine residues separated by more than 200 residues are held in close proximity to each other on adjacent barrel strands.

Identification and characterization of the acoD gene encoding a dihydrolipoamide dehydrogenase of the Klebsiella pneumoniae acetoin dehydrogenase system.

The acoD gene, which encodes a dihydrolipoamide dehydrogenase component of the acetoin dehydrogenase enzyme system of Klebsiella pneumoniae was isolated and the nucleotide sequence determined. The gene is capable of encoding a protein of 465 amino acid residues with conserved binding domains for NAD and FAD, and two redox-active cysteine residues. The acoD gene product exhibited a Michaelis constant of 170 microM for NAD, while NADP can not be used as a substrate. The purified enzyme appeared to be a dimer of the acoD gene product. It did not associate tightly with the E1 and E2 components of either acetoin dehydrogenase or 2-oxoglutarate dehydrogenase to form an active multi-enzyme complex.

Crystallographic investigations of ribonucleotide reductase

Introduction Ribonucleotide reductase catalyses the de novo production of deoxyribonucleotides. The enzyme reduces all the four main ribonucleotides to the corresponding deoxyribonucleotides. In higher organisms and in Eschmkhziz coli. this takes place at the diphosphate level I 1 1. Kibonucleotide reductase from E. coZi is a multisubunit alpL enzyme, where the two homodinieric proteins are denoted R1 and R2. The larger a L subunit I i l contains the binding sites for the substrate and for allosteric effectors, and also the redox-active cysteine residues [ 1 J. Subunit R1 has a molecular mass of 2 x 86 k1)a and each subunit contains 76 1 residues. K 1 carries two types of allosteric-effector sites: an overall activity site and a substrate-specificity site. ATP works as a general effector for eilzynie activity. The substrate-specificity effectors regulates the enzyme, so that suitable proportions of the different deoxyribonucleotides are produced. The smaller pL protein, denoted R2, contains the dinuclear ferric centre, and a stable free tyrosyl radical that is necessary for the enzymic activity [ 21. l'he K2 protein has a molecular mass of 2x43,s k l h , and each subunit contains 375 amino acids. K 2 was shown to contain a tyrosyl radical a long time ago, and the radical was shown to be localized over the tyrosine ring. From sequence comparisons, the radical tyrosine was suggested to be Tyr', which was later confirmed by site-directed mutagenesis 13). 7'he radical is generated in close cooperation with a binuclear iron centre by molecular oxygen. T h e ribonucleotide subunit K 2 was the first enzyme for which a stable free protein radical was ch;iracterized [ 21. Since then, a number of enzymes have been shown to use amino acids as 'built in' redox cofactors in radical-based reactions (see other papers in this colloquium). 'l'he radical tyrosyl122 is essential for the activity of the enzyme, and was therefore thought to participate in the enzymic reaction by creating an intermediate substrate radical, thereby activating the substrate for further reduction by a pair of thiols [4]. The tyrosyl radical is not consumed in the substrate-reduction cycle and the radical transfer between Tyr' and the substrate must be reversible in such a scheme. The tyrosyl radical can be scavenged by different nucleotide analogues binding at the active site, which demonstrates the feasibility of a radical transfer from Tyr* to the substrate. There is, however, no evidence yet for a radical transfer in the other direction, from the substrate to Tyr'.

Identification and characterization of an Escherichia coli gene required for the formation of correctly folded alkaline phosphatase, a periplasmic enzyme.

Tn5 insertion mutations of Escherichia coli were isolated that impaired the formation of correctly folded alkaline phosphatase (PhoA) in the periplasm. The PhoA polypeptide synthesized in the mutants was translocated across the cytoplasmic membrane but not released into the periplasmic space. It was susceptible to degradation by proteases in vivo and in vitro. The wild-type counterpart of this gene (named ppfA) has been sequenced and shown to encode a periplasmic protein with a pair of potentially redox-active cysteine residues. PhoA synthesized in the mutants indeed lacked disulfide bridges. These results indicate that the folding of PhoA in vivo is not spontaneous but catalyzed at least at the disulfide bond formation step.

Evidence for two different classes of redox-active cysteines in ribonucleotide reductase of Escherichia coli

The large subunit of ribonucleotide reductase from Escherichia coli contains redox-active cysteine residues. In separate experiments, five conserved and 2 nonconserved cysteine residues were substituted with alanines by oligonucleotide-directed mutagenesis. The activities of the mutant proteins were determined in the presence of three different reductants: thioredoxin, glutaredoxin, or dithiothreitol. The results indicate two different classes of redox-active cysteines in ribonucleotide reductase: 1) C-terminal Cys-754 and Cys-759 responsible for the interaction with thioredoxin and glutaredoxin; and 2) Cys-225 and Cys-439 located at the nucleotide-binding site. Our classification of redox-active cysteines differs from the location of the active site cysteines in E. coli ribonucleotide reductase suggested previously (Lin, A.-N. I., Ashley, G. W., and Stubbe, J. (1987) Biochemistry 26, 6905-6909).