Pyruvate formate lyase is structurally homologous to type I ribonucleotide reductase.

BACKGROUND: Pyruvate formate lyase (PFL) catalyses a key step in Escherichia coli anaerobic glycolysis by converting pyruvate and CoA to formate and acetylCoA. The PFL mechanism involves an unusual radical cleavage of pyruvate, involving an essential C alpha radical of Gly734 and two cysteine residues, Cys418 and Cys419, which may form thiyl radicals required for catalysis. We undertook this study to understand the structural basis for catalysis. RESULTS: The first structure of a fragment of PFL (residues 1-624) at 2.8 A resolution shows an unusual barrel-like structure, with a catalytic beta finger carrying Cys418 and Cys419 inserted into the centre of the barrel. Several residues near the active-site cysteines can be ascribed roles in the catalytic mechanism: Arg176 and Arg435 are positioned near Cys419 and may bind pyruvate/formate and Trp333 partially buries Cys418. Both cysteine residues are accessible to each other owing to their cis relationship at the tip of the beta finger. Finally, two clefts that may serve as binding sites for CoA and pyruvate have been identified. CONCLUSIONS: PFL has striking structural homology to the aerobic ribonucleotide reductase (RNR): the superposition of PFL and RNR includes eight of the ten strands in the unusual RNR alpha/beta barrel as well as the beta finger, which carries key catalytic residues in both enzymes. This provides the first structural proof that RNRs and PFLs are related by divergent evolution from a common ancestor.     

Inactivation of pyruvate formate-lyase by dioxygen: defining the mechanistic interplay of glycine 734 and cysteine 419 by rapid freeze-quench EPR

Pyruvate formate-lyase from Escherichia coli (EC 2.3.1.54; PFL) catalyzes the reversible anaerobic conversion of pyruvate and CoA into acetyl-CoA and formate. Active PFL contains a novel alpha-carbon centered glycyl radical at G734 that is required for its catalytic activity. Two adjacent cysteine residues, C418 and C419, are essential for PFL activity according to site-directed mutagenesis studies. Upon exposure to air, active PFL loses its activity with the concomitant loss of the glycyl radical. Previous EPR studies of dioxygen inactivation of PFL revealed protein-based peroxyl and sulfinyl radicals during the manual mixing and quenching process [Reddy et al. (1998) Biochemistry 37, 558-563]. To probe the mechanism of this process, we carried out experiments using rapid freeze-quench EPR spectroscopy. Upon mixing of active wild type or C418A PFL with oxygenated solution, a short-lived radical intermediate appears at the earliest time point (10 ms), followed by the appearance of a long-lived sulfinyl radical. The axial EPR spectrum of this short-lived radical (g = 2.034, 2.007) is characteristic of a peroxyl radical. When C419A PFL or the double mutant [C418A/C419A] PFL was mixed with oxygenated solution, the peroxyl radical was also observed at 10 ms but in this case persisted over 12 s. These observations provide compelling evidence to support a proposed mechanism in which dioxygen quenches the glycyl radical in the active enzyme and the resulting peroxyl radical may react further with the sulfhydryl group of the C419 residue to form the sulfinyl radical.     

Modification of Cys-418 of pyruvate formate-lyase by methacrylic acid, based on its radical mechanism

The recently determined crystal structure of pyruvate formate-lyase (PFL) suggested a new view of the mechanism of this glycyl radical enzyme, namely that intermediary thiyl radicals of Cys-418 and Cys-419 participate in different ways [Becker, A. et al. (1999) Nat. Struct. Biol. 6, 969-975]. We report here a suicide reaction of PFL that occurs with the substrate-analog methacrylate with retention of the protein radical (K(I)=0.42 mM, k(i)=0.14 min(-1)). Using [1-(14)C]methacrylate (synthesized via acetone cyanhydrin), the reaction end-product was identified by peptide mapping and cocrystallization experiments as S-(2-carboxy-(2S)-propyl) substituted Cys-418. The stereoselectivity of the observed Michael addition reaction is compatible with a radical mechanism that involves Cys-418 thiyl as nucleophile and Cys-419 as H-atom donor, thus supporting the functional assignments of these catalytic amino acid residues derived from the protein structure.     

YfiD of Escherichia coli and Y06I of bacteriophage T4 as autonomous glycyl radical cofactors reconstituting the catalytic center of oxygen-fragmented pyruvate formate-lyase.

Reaction of oxygen with the glycyl radical in pyruvate formate-lyase (PFL) leads to cleavage of the polypeptide backbone between N-Calpha of Gly734. A recombinant protein comprising the core of PFL (Ser1-Ser733) is shown here to associate with the YfiD protein (14 kDa) of Escherichia coli and likewise with the homologous T4 encoded Y06I protein, yielding upon reaction with PFL activase a heterooligomeric PFL enzyme that has full catalytic activity (35 U/nmol). Treatment of the activated complexes with oxygen led to cleavage of the 14 kDa proteins into 11 and 3 kDa polypeptides as expected for the localization of the putative glycyl radical at Gly102 (YfiD) or Gly95 (Y06I). For the isolated fragments from Y06I, mass spectrometric analysis (nanoESI-MS) determined a C-terminal serine carboxamide in the 11 kDa fragment, and a N-terminal oxalyl modification in the 3 kDa fragment. We speculate that YfiD in E. coli and other facultative anaerobic bacteria has evolved as a "spare part" for PFL's glycyl radical domain, utilized for rapid recovery of PFL activity (and thus ATP generation) in cells that have experienced oxidative stress.     

Pyruvate Formate-lyase,Evidence for an Open Conformation Favored in the Presence of Its Activating Enzyme

Pyruvate formate-lyase-activating enzyme (PFL-AE) activates pyruvate formate-lyase (PFL) by generating a catalytically essential radical on Gly-734 of PFL. Crystal structures of unactivated PFL reveal that Gly-734 is buried 8 Å from the surface of the protein in what we refer to here as the closed conformation of PFL. We provide here the first experimental evidence for an alternate open conformation of PFL in which: (i) the glycyl radical is significantly less stable; (ii) the activated enzyme exhibits lower catalytic activity; (iii) the glycyl radical undergoes less H/D exchange with solvent; and (iv) the T_m of the protein is decreased. The evidence suggests that in the open conformation of PFL, the Gly-734 residue is located not in its buried position in the enzyme active site but rather in a more solvent-exposed location. Further, we find that the presence of the PFL-AE increases the proportion of PFL in the open conformation; this observation supports the idea that PFL-AE accesses Gly-734 for direct hydrogen atom abstraction by binding to the Gly-734 loop in the open conformation, thereby shifting the closed ↔ open equilibrium of PFL to the right. Together, our results lead to a model in which PFL can exist in either a closed conformation, with Gly-734 buried in the active site of PFL and harboring a stable glycyl radical, or an open conformation, with Gly-734 more solvent-exposed and accessible to the PFL-AE active site. The equilibrium between these two conformations of PFL is modulated by the interaction with PFL-AE.     

Intramolecular electron transfer between tyrosyl radical and cysteine residue inhibits tyrosine nitration and induces thiyl radical formation in model peptides treated with myeloperoxidase, H2O2, and NO2-: EPR SPIN trapping studies

We investigated the effects of a cysteine residue on tyrosine nitration in several model peptides treated with myeloperoxidase (MPO), H(2)O(2), and nitrite anion (NO(2)(-)) and with horseradish peroxidase and H(2)O(2). Sequences of model peptides were acetyl-Tyr-Cys-amide (YC), acetyl-Tyr-Ala-Cys-amide (YAC), acetyl-Tyr-Ala-Ala-Cys-amide (YAAC), and acetyl-Tyr-Ala-Ala-Ala-Ala-Cys-amide (YAAAAC). Results indicate that nitration and oxidation products of tyrosyl residue in YC and other model peptides were barely detectable. A major product detected was the corresponding disulfide (e.g. YCysCysY). Spin trapping experiments with 5,5'-dimethyl-1-pyrroline N-oxide (DMPO) revealed thiyl adduct (e.g. DMPO-SCys-Tyr) formation from peptides (e.g. YC) treated with MPO/H(2)O(2) and MPO/H(2)O(2)/NO(2)(-). The steady-state concentrations of DMPO-thiyl adducts decreased with increasing chain length of model peptides. Blocking the sulfydryl group in YC with methylmethanethiosulfonate (that formed YCSSCH(3)) totally inhibited thiyl radical formation as did substitution of Tyr with Phe (i.e. FC) in the presence of MPO/H(2)O(2)/NO(2)(-). However, increased tyrosine nitration, tyrosine dimerization, and tyrosyl radical formation were detected in the MPO/H(2)O(2)/NO(2)(-)/YCSSCH(3) system. Increased formation of S-nitrosated YC (YCysNO) was detected in the MPO/H(2)O(2)/(*)NO system. We conclude that a rapid intramolecular electron transfer reaction between the tyrosyl radical and the Cys residue impedes tyrosine nitration and induces corresponding thiyl radical and nitrosocysteine product. Implications of this novel intramolecular electron transfer mechanism in protein nitration and nitrosation are discussed.     

Two active site asparagines are essential for the reaction mechanism of the class III anaerobic ribonucleotide reductase from bacteriophage T4

Class III ribonucleotide reductase is an anaerobic enzyme that uses a glycyl radical to catalyze the reduction of ribonucleotides to deoxyribonucleotides and formate as ultimate reductant. The reaction mechanism of class III ribonucleotide reductases requires two cysteines within the active site, Cys-79 and Cys-290 in bacteriophage T4 NrdD numbering. Cys-290 is believed to form a transient thiyl radical that initiates the reaction with substrate and Cys-79 to take part as a transient thiyl radical in later steps of the reductive reaction. The recently solved three-dimensional structure of class III ribonucleotide reductase (RNR) from bacteriophage T4 shows that two highly conserved asparagines, Asn-78 and Asn-311, are positioned close to the essential Cys-79. We have investigated the function of Asn-78 and Asn-311 by site-directed mutagenesis and measured enzyme activity and glycyl radical formation in five single (N78(A/C/D) and N311(A/C)) and one double (N78A/N311A) mutant proteins. Our results suggest that both asparagines are important for the catalytic mechanism of class III RNR and that one asparagine can partially compensate for the lack of the other functional group in the single Asn --> Ala mutant proteins. A plausible role for these two asparagines could be in positioning formate in the active site to orient it toward the proposed thiyl radical of Cys-79. This would also control the highly reactive carbon dioxide radical anion form of formate within the active site before it is released as carbon dioxide. A detailed reaction scheme including the function of the two asparagines and two formate molecules is proposed for class III RNRs.     

Pyruvate formate-lyase-activating enzyme: strictly anaerobic isolation yields active enzyme containing a [3Fe-4S](+) cluster

Pyruvate formate-lyase-activating enzyme (PFL-AE) from Escherichia coli (E. coli) catalyzes the stereospecific abstraction of a hydrogen atom from Gly734 of pyruvate formate-lyase (PFL) in a reaction that is strictly dependent on the cosubstrate S-adenosyl-l-methionine (AdoMet). Although PFL-AE is an iron-dependent enzyme, isolation of the enzyme with its metal center intact has proven difficult due to the oxygen sensitivity and lability of the metal center. We report here the first isolation of PFL-AE under nondenaturing, strictly anaerobic conditions. Iron and sulfide analysis as well as UV-visible, EPR, and resonance Raman data support the presence of a [3Fe-4S](+) cluster in the purified enzyme. The isolated native enzyme, but not apo-enzyme, exhibits a high specific activity (31 U/mg) in the absence of added iron, indicating that the native cluster is necessary and sufficient for enzymatic activity.     

S4 charges move close to residues in the pore domain during activation in a K channel.

Voltage-gated ion channels respond to changes in the transmembrane voltage by opening or closing their ion conducting pore. The positively charged fourth transmembrane segment (S4) has been identified as the main voltage sensor, but the mechanisms of coupling between the voltage sensor and the gates are still unknown. Obtaining information about the location and the exact motion of S4 is an important step toward an understanding of these coupling mechanisms. In previous studies we have shown that the extracellular end of S4 is located close to segment 5 (S5). The purpose of the present study is to estimate the location of S4 charges in both resting and activated states. We measured the modification rates by differently charged methanethiosulfonate regents of two residues in the extracellular end of S5 in the Shaker K channel (418C and 419C). When S4 moves to its activated state, the modification rate by the negatively charged sodium (2-sulfonatoethyl) methanethiosulfonate (MTSES(-)) increases significantly more than the modification rate by the positively charged [2-(trimethylammonium)ethyl] methanethiosulfonate, bromide (MTSET(+)). This indicates that the positive S4 charges are moving close to 418C and 419C in S5 during activation. Neutralization of the most external charge of S4 (R362), shows that R362 in its activated state electrostatically affects the environment at 418C by 19 mV. In contrast, R362 in its resting state has no effect on 418C. This suggests that, during activation of the channel, R362 moves from a position far away (>20 A) to a position close (8 A) to 418C. Despite its close approach to E418, a residue shown to be important in slow inactivation, R362 has no effect on slow inactivation or the recovery from slow inactivation. This refutes previous models for slow inactivation with an electrostatic S4-to-gate coupling. Instead, we propose a model with an allosteric mechanism for the S4-to-gate coupling.     

3'-Phosphoadenosine-5'-phosphosulfate reductase in complex with thioredoxin: a structural snapshot in the catalytic cycle

The crystal structure of Escherichia coli 3'-phosphoadenosine-5'-phosphosulfate (PAPS) reductase in complex with E. coli thioredoxin 1 (Trx1) has been determined to 3.0 A resolution. The two proteins are covalently linked via a mixed disulfide that forms during nucleophilic attack of Trx's N-terminal cysteine on the Sgamma atom of the PAPS reductase S-sulfocysteine (E-Cys-Sgamma-SO3-), a central intermediate in the catalytic cycle. For the first time in a crystal structure, residues 235-244 in the PAPS reductase C-terminus are observed, depicting an array of interprotein salt bridges between Trx and the strictly conserved glutathione-like sequence, Glu238Cys239Gly240Leu241His242. The structure also reveals a Trx-binding surface adjacent to the active site cleft and regions of PAPS reductase associated with conformational change. Interaction at this site strategically positions Trx to bind the S-sulfated C-terminus and addresses the mechanism for requisite structural rearrangement of this domain. An apparent sulfite-binding pocket at the protein-protein interface explicitly orients the S-sulfocysteine Sgamma atom for nucleophilic attack in a subsequent step. Taken together, the structure of PAPS reductase in complex with Trx highlights the large structural rearrangement required to accomplish sulfonucleotide reduction and suggests a role for Trx in catalysis beyond the paradigm of disulfide reduction.     

Is Arg418 the catalytic base required for the hydrolysis step of the IMP dehydrogenase reaction?

The first committed step of guanine nucleotide biosynthesis is the oxidation of inosine 5'-monophosphate (IMP) to xanthosine 5'-monophosphate (XMP) catalyzed by IMP dehydrogenase. The reaction involves the reduction of NAD(+) with the formation of a covalent enzyme intermediate (E-XMP). Hydrolysis of E-XMP requires the enzyme to adopt a closed conformation and is rate-limiting. Thr321, Arg418, and Tyr419 are candidates for the residue that activates water. The substitution of Thr321 has similar, but small, effects on both the hydride transfer and hydrolysis steps. This result suggests that Thr321 influences the reactivity of Cys319, either through a direct interaction or by stabilizing the structure of the active site loop. The hydrolysis of E-XMP is accelerated by the deprotonation of a residue with a pK(a) of approximately 8. A similar deprotonation stabilizes the closed conformation; this residue has a pK(a) of >or=6 in the closed conformation. The substitution of Tyr419 with Phe does not change the pH dependence of either the hydrolysis of E-XMP or the conformational change, which suggests that Tyr419 is not the residue that activates water. In contrast, the conformational change becomes pH-independent when Arg418 is substituted with Gln. Lys can replace the function of Arg418 in the hydrolysis reaction but does not stabilize the closed conformation. The simplest explanation for these observations is that Arg418 serves as the base that activates water in the IMPDH reaction.     

Computer-aided design of the stability of pyruvate formate-lyase from Escherichia coli by site-directed mutagenesis

Using computer-aided design of single-site mutations, three amino acid residues determined by changes in folding free energy between wild-type (wt) and mutant proteins were exchanged to enhance the stability of pyruvate formate-lyase (PFL). The mutant enzymes were tested for properties such as optimum temperature, optimum pH, kinetic parameters, and stability to temperature. There were two mutant variants, Glu336Cys and Glu400Ile, that exhibited increased thermostability as compared to the wt enzyme. The melting temperatures (T(m), the temperature at which 50% inactivation occurs after heat treatment for 20 min) of Glu336Cys and Glu400Ile increased by 3.7 and 2.2 respectively. They also showed an increase in half life of about 1.80 and 2.21-fold, whereas Ala273Cys showed a slight decrease as compared with the wt enzyme.     

Pyruvate Formate-lyase and Its Activation by Pyruvate Formate-lyase Activating Enzyme

The activation of pyruvate formate-lyase (PFL) by pyruvate formate-lyase activating enzyme (PFL-AE) involves formation of a specific glycyl radical on PFL by the PFL-AE in a reaction requiring S-adenosylmethionine (AdoMet). Surface plasmon resonance experiments were performed under anaerobic conditions on the oxygen-sensitive PFL-AE to determine the kinetics and equilibrium constant for its interaction with PFL. These experiments show that the interaction is very slow and rate-limited by large conformational changes. A novel AdoMet binding assay was used to accurately determine the equilibrium constants for AdoMet binding to PFL-AE alone and in complex with PFL. The PFL-AE bound AdoMet with the same affinity (∼6 μm) regardless of the presence or absence of PFL. Activation of PFL in the presence of its substrate pyruvate or the analog oxamate resulted in stoichiometric conversion of the [4Fe-4S]¹⁺ cluster to the glycyl radical on PFL; however, 3.7-fold less activation was achieved in the absence of these small molecules, demonstrating that pyruvate or oxamate are required for optimal activation. Finally, in vivo concentrations of the entire PFL system were calculated to estimate the amount of bound protein in the cell. PFL, PFL-AE, and AdoMet are essentially fully bound in vivo, whereas electron donor proteins are partially bound.     

Structure and function of transmembrane segment XII in osmosensor and osmoprotectant transporter ProP of Escherichia coli

Escherichia coli transporter ProP acts as both an osmosensor and an osmoregulator. As medium osmolality rises, ProP is activated and mediates H+-coupled uptake of osmolytes like proline. A homology model of ProP with 12-transmembrane (TM) helices and cytoplasmic termini was created, and the protein's topology was substantiated experimentally. Residues 468-497, at the end of the C-terminal domain and linked to TM XII, form an intermolecular, homodimeric alpha-helical coiled-coil that tunes the transporter's response to osmolality. We aim to further define the structure and function of ProP residues Q415-E440, predicted to include TM XII. Each residue was replaced with cysteine (Cys) in a histidine-tagged, Cys-less ProP variant (ProP*). Cys at positions 415-418 and 438-440 were most reactive with Oregon Green Maleimide (OGM), suggesting that residues 419 through 437 are in the membrane. Except for V429-I433, reactivity of those Cys varied with helical periodicity. Cys predicted to face the interior of ProP were more reactive than Cys predicted to face the lipid. The former may be exposed to hydrated polar residues in the protein interior, particularly on the periplasmic side. Intermolecular cross-links formed when ProP* variants with Cys at positions 419, 420, 422, and 439 were treated with DTME. Thus TM XII can participate, along its entire length, in the dimer interface of ProP. Cys substitution E440C rendered ProP* inactive. All other variants retained more than 30% of the proline uptake activity of ProP* at high osmolality. Most variants with Cys substitutions in the periplasmic half of TM XII activated at lower osmolalities than ProP*. Variants with Cys substitutions on one face of the cytoplasmic half of TM XII required a higher osmolality to activate. They included elements of a GXXXG motif that are predicted to form the interface of TM XII with TM VII. These studies define the position of ProP TM XII within the membrane, further support the predicted structure of ProP, reveal the dimerization interface, and show that the structure of TM XII influences the osmolality at which ProP activates.     

Substitution of the conserved Arg-Tyr dyad selectively disrupts the hydrolysis phase of the IMP dehydrogenase reaction

Inosine 5'-monophosphate dehydrogenase (IMPDH) catalyzes the oxidation of IMP to XMP via the covalent E-XMP* intermediate (E-XMP*), with the concomitant reduction of NAD(+). Hydrolysis of E-XMP* is rate-limiting, and the catalytic base required for this step has not been identified. An X-ray crystal structure of Tritrichomonas foetus IMPDH with mizoribine monophosphate (MZP) reveals a novel closed conformation in which a mobile flap occupies the NAD(+)/NADH site [Gan, L., Seyedsayamdost, M. R., Shuto, S., Matsuda, A., Petsko, G. A., and Hedstrom, L. (2003) Biochemistry 42, 857-863]. In this complex, a water molecule is coordinated between flap residues Arg418 and Tyr419 and MZP in a geometry that resembles the transition state for hydrolysis of E-XMP*, which suggests that the Arg418-Tyr419 dyad activates water. We constructed and characterized two point mutants, Arg418Ala and Tyr419Phe, to probe the role of the Arg418-Tyr419 dyad in the IMPDH reaction. Arg418Ala and Tyr419Phe decrease k(cat) by factors of 500 and 10, respectively, but have no effect on hydride transfer or NADH release. In addition, the mutants display increased solvent isotope effects and increased levels of steady-state accumulation of E-XMP*. Inhibitor analysis indicates that the mutations destabilize the closed conformation, but this effect can account for a decrease in k(cat) of no more than a factor of 2. These observations demonstrate that both the Arg418Ala and Tyr419Phe mutations selectively impair hydrolysis of E-XMP* by disrupting the chemical transformation. Moreover, since the effects of the Tyr419Phe mutation are comparatively small, these experiments suggest that Arg418 acts as the base to activate water.     

Computer-Aided Design of the Stability of Pyruvate Formate-Lyase from Escherichia coli by Site-Directed Mutagenesis

Using computer-aided design of single-site mutations, three amino acid residues determined by changes in folding free energy between wild-type (wt) and mutant proteins were exchanged to enhance the stability of pyruvate formate-lyase (PFL). The mutant enzymes were tested for properties such as optimum temperature, optimum pH, kinetic parameters, and stability to temperature. There were two mutant variants, Glu336Cys and Glu400Ile, that exhibited increased thermostability as compared to the wt enzyme. The melting temperatures (T _m, the temperature at which 50% inactivation occurs after heat treatment for 20 min) of Glu336Cys and Glu400Ile increased by 3.7 and 2.2 respectively. They also showed an increase in half life of about 1.80 and 2.21-fold, whereas Ala273Cys showed a slight decrease as compared with the wt enzyme.     

Crystal structure of a glycyl radical enzyme from Archaeoglobus fulgidus

We have solved the crystal structure of a PFL2 from Archaeglobus fulgidus at 2.9 A resolution. Of the three previously solved enzyme structures of glycyl radical enzymes, pyruvate formate lyase (PFL), anaerobic ribonucleotide reductase and glycerol dehydratase (GD), the last one is clearly most similar to PFL2. We observed electron density in the active site of PFL2, which we modelled as glycerol. The orientation of the glycerol is different from that in GD, and changes in the active site indicate that the actual substrate of PFL2 is bigger than a glycerol molecule, but sequence and structural homology suggest that PFL2 may be a dehydratase. Crystal packing, solution X-ray scattering and ultracentrifugation experiments show that PFL2 is tetrameric, unlike other glycyl radical enzymes. A.fulgidus is a hyperthermophile and PFL2 appears to be stabilized by several factors including an increased number of ion pairs, differences in buried charges, a truncated N terminus, anchoring of loops and N terminus via salt-bridges, changes in the oligomeric interface and perhaps also the higher oligomerization state of the protein.     

Evidence for a free radical mechanism of styrene-glutathione conjugate formation catalyzed by prostaglandin H synthase and horseradish peroxidase

We have proposed, using styrene as a model, a new mechanism for the formation of glutathione conjugates that is independent of epoxide formation but dependent on the oxidation of glutathione to a thiyl radical by peroxidases such as prostaglandin H synthase or horseradish peroxidase. The thiyl radical reacts with styrene to yield a carbon-centered radical which subsequently reacts with molecular oxygen to give the styrene-glutathione conjugate. We have used electron spin resonance spin trapping techniques to detect the proposed free radical intermediates. A styrene carbon-centered radical was trapped using the spin traps 5,5-dimethyl-1-pyrroline N-oxide (DMPO) and t-nitrosobutane. The position of the carbon-centered radical was confirmed to be at carbon 7 by the use of specific 2H-labeled styrenes. The addition of the spin trap DMPO inhibited both the utilization of molecular oxygen and the formation of styrene-glutathione conjugates. Under anaerobic conditions additional styrene-glutathione conjugates were formed, one of which was identified by fast atom bombardment mass spectrometry as S-(2-phenyl)ethylglutathione. The glutathione thiyl radical intermediate was observed by spin trapping with DMPO. These results support the proposed free radical-mediated formation of styrene-glutathione conjugates by peroxidase enzymes.     

Unpaired Electron Migration between Aromatic and Sulfur Peptide Units

Cysteine thiyl radicals (Cys/S') were found capable of one-electron oxidation of tyrosine. Equilibration occurred, using Cys and Gly-Tyr, with an equilibrium constant of K₅ = 20 ± 4 at pH 9.15: Cys/S- + Tyr = Cys + Tyr/O

Hence the reduction potentials of these couples differ at pH 9.15 by E(Cys/S', Cys) - E(Tyr/O^r, Tyr) = 80 mV. Oxidation of Trp-Gly by Cys/S' was not detectable from pH 7 to 12. The methionyl radical cation (Met/S'N), formed via 'OH-attack on Met-Gly, reacts with Trp-Gly to generate the indolyl radical (Trp/N'). New results on intramolecular Trp/N' → Tyr/O' transitions indicate that the reaction requires direct contact between the two redox centers. Various possible pathways for migration of unpaired electrons between peptide units are compiled in a scheme.     

Catalytic-site mapping of pyruvate formate lyase. Hypophosphite reaction on the acetyl-enzyme intermediate affords carbon-phosphorus bond synthesis (1-hydroxyethylphosphonate)

Pyruvate formate-lyase of Escherichia coli cells, a homodimeric protein of 2 x 85 kDa, is distinguished by the property of containing a stable organic free radical (g = 2.0037) in its resting state. The enzyme (E-SH) achieves pyruvate conversion to acetyl-CoA via two distinct half-reactions (E-SH + pyruvate in equilibrium E-S-acetyl + formate; E-S-acetyl + CoA in equilibrium E-SH + acetyl-CoA), the first of which has been proposed to involve reversible homolytic carbon-carbon bond cleavage [J. Knappe et al. (1984) Proc. Natl Acad. Sci. USA 81, 1332-1335]. Present studies identified Cys-419 as the covalent-catalytic cysteinyl residue via CNBr fragmentation of E-S-[14C]acetyl and radio-sequencing of the isolated peptide CB-Ac (amino acid residues 406-423). Reaction of the formate analogue hypophosphite with E-S-acetyl was investigated and found to produce 1-hydroxyethylphosphonate with a thioester linkage to the adjacent Cys-418. The structure was determined from the chymotryptic peptide CH-P (amino acid residues 415-425), using 31P-NMR spectroscopy (delta = 44 ppm) and by chemical characterisation through degradation into 1-hydroxyethylphosphonate with phosphodiesterase or bromine. This novel P-C-bond synthesis involves the enzyme-based free radical and is proposed to resemble the physiological C-C-bond synthesis (pyruvate production) from formate and E-S-acetyl. These findings are interpreted as proof of a radical mechanism for the action of pyruvate formate-lyase. The central Cys-418/Cys-419 pair of the active site shows a distinctive thiolate property even in the inactive (nonradical) form of the enzyme, as determined using an iodoacetate probe.