David P. Ballou

Oxidative Protein Folding Is Driven by the Electron Transport System

Disulfide bond formation is catalyzed in vivo by DsbA and DsbB. Here we reconstitute this oxidative folding system using purified components. We have found the sources of oxidative power for protein folding and show how disulfide bond formation is linked to cellular metabolism. We find that disulfide bond formation and the electron transport chain are directly coupled. DsbB uses quinones as electron acceptors, allowing various choices for electron transport to support disulfide bond formation. Electrons flow via cytochrome bo oxidase to oxygen under aerobic conditions or via cytochrome bd oxidase under partially anaerobic conditions. Under truly anaerobic conditions, menaquinone shuttles electrons to alternate final electron acceptors such as fumarate. This flexibility reflects the vital nature of the disulfide catalytic system.

Direct demonstration of superoxide anion production during the oxidation of reduced flavin and of its catalytic decomposition by erythrocuprein

The oxidation of reduced flavins by molecular oxygen at neutral to alkaline pH produces substantial yields of the superoxide anion, O2•. This species is rapidly destroyed by catalytic quantities of the copper protein, erythrocuprein, and by stoichiometric quantities of ferricytochrome c.

Spectral intermediates in the reaction of oxygen with purified liver microsomal cytochrome P-450.

Abstract Stopped flow spectrophotometry has shown the occurrence of two distinct spectral intermediates in the reaction of oxygen with the reduced form of highly purified cytochrome P-450 from liver microsomes. As indicated by difference spectra, Complex I (with maxima at 430 and 450 nm) is rapidly formed and then decays to form Complex II (with a broad maximum at 440 nm), which resembles the intermediate seen in steady state experiments. In the reaction sequence, P-450LMred → O 2 Complex I→Complex II→P-450LMox the last step is rate-limiting. The rate of that step is inadequate to account for the known turnover number of the enzyme in benzphetamine hydroxylation unless NADPH-cytochrome P-450 reductase or cytochrome b 5 is added. The latter protein does not appear to function as an electron carrier in this process.

Revisiting the kinetics of nitric oxide (NO) binding to soluble guanylate cyclase: The simple NO-binding model is incorrect

Soluble guanylate cyclase (sGC) is a ferrous iron hemoprotein receptor for nitric oxide (NO). NO binding to the heme activates the enzyme 300-fold. sGC as isolated is five-coordinate, ferrous with histidine as the axial ligand. The NO-activated enzyme is a five-coordinate nitrosyl complex where the axial histidine bond is broken. Past studies using rapid-reaction kinetics demonstrated that both the formation of a six-coordinate intermediate and the conversion of the intermediate to the activated five-coordinate nitrosyl complex depended on the concentration of NO. A model invoking a second NO molecule as a catalyst for the conversion of the six-coordinate intermediate to the five-coordinate sGC–NO complex was proposed to explain the observed kinetic data. A recent study [Bellamy, T. C., Wood, J. & Garthwaite, J. (2002) Proc. Natl. Acad. Sci. USA 99, 507–510] concluded that a simple two-step binding model explains the results. Here we show through further analysis and simulations of previous data that the simple two-step binding model cannot be used to describe our results. Instead we show that a slightly more complex two-step binding model, where NO is used as a ligand in the first step and a catalyst in the second step, can describe our results quite satisfactorily. These new simulations combined with the previous activation data lead to the conclusion that the intermediate six-coordinate sGC–NO complex has substantial activity. The model derived from our simulations also can account for the slow deactivation of sGC that has been observed in vitro.

The biochemical mechanism of auxin biosynthesis by an Arabidopsis YUCCA flavin-containing monooxygenase

Background: Auxin is essential for plant growth, but its biosynthesis in plants has not been biochemically defined. Results: Key features of the catalytic mechanism for the YUCCA flavoprotein, the rate-limiting enzyme of auxin biosynthesis, are determined. Conclusion: YUCs generate an observable though relatively short lived C4a-(hydro)peroxyflavin intermediate for catalysis in auxin biosynthesis. Significance: This work establishes the previously unknown biochemical mechanism of auxin biosynthesis. Auxin regulates every aspect of plant growth and development. Previous genetic studies demonstrated that YUCCA (YUC) flavin-containing monooxygenases (FMOs) catalyze a rate-limiting step in auxin biosynthesis and that YUCs are essential for many developmental processes. We proposed that YUCs convert indole-3-pyruvate (IPA) to indole-3-acetate (IAA). However, the exact biochemical mechanism of YUCs has remained elusive. Here we present the biochemical characterization of recombinant Arabidopsis YUC6. Expressed in and purified from Escherichia coli, YUC6 contains FAD as a cofactor, which has peaks at 448 nm and 376 nm in the UV-visible spectrum. We show that YUC6 uses NADPH and oxygen to convert IPA to IAA. The first step of the YUC6-catalyzed reaction is the reduction of the FAD cofactor to FADH− by NADPH. Subsequently, FADH− reacts with oxygen to form a flavin-C4a-(hydro)peroxy intermediate, which we show has a maximum absorbance at 381 nm in its UV-visible spectrum. The final chemical step is the reaction of the C4a-intermediate with IPA to produce IAA. Although the sequences of the YUC enzymes are related to those of the mammalian FMOs, which oxygenate nucleophilic substrates, YUC6 oxygenates an electrophilic substrate (IPA). Nevertheless, both classes of enzymes form quasi-stable C4a-(hydro)peroxyl FAD intermediates. The YUC6 intermediate has a half-life of ∼20 s whereas that of some FMOs is >30 min. This work reveals the catalytic mechanism of the first known plant flavin monooxygenase and provides a foundation for further investigating how YUC activities are regulated in plants.

Kinetic Analysis of the Catalytic Domain of Human Cdc25B

The Cdc25 cell cycle regulator is a member of the dual-specificity class of protein-tyrosine phosphatases that hydrolyze phosphotyrosine- and phosphothreonine-containing substrates. To study the mechanism of Cdc25B, we have overexpressed and purified the catalytic domain of human Cdc25B (Xu, X., and Burke, S. P. (1996) J. Biol. Chem. 271, 5118-5124). In the present work, we have analyzed the kinetic properties of the Cdc25B catalytic domain using the artificial substrate 3-O-methylfluorescein phosphate (OMFP). Steady-state kinetic analysis indicated that the kcat/Km for OMFP hydrolysis is almost 3 orders of magnitude greater than that for p-nitrophenyl phosphate hydrolysis. Like other dual-specificity phosphatases, Cdc25 exhibits a two-step catalytic mechanism, characterized by formation and breakdown of a phosphoenzyme intermediate. Pre-steady-state kinetic analysis of OMFP hydrolysis indicated that formation of the phosphoenzyme intermediate is ∼20 times faster than subsequent phosphoenzyme breakdown. The resulting burst pattern of product formation allowed us to derive rate constants for enzyme phosphorylation (26 s−1) and dephosphorylation (1.5 s−1) as well as the dissociation constant for OMFP (0.3 mM). Calculations suggest that OMFP binds with higher affinity and reacts faster with Cdc25B than does p-nitrophenyl phosphate. OMFP is a highly efficient substrate for the dual-specificity protein-tyrosine phosphatases VHR and rVH6, but not for two protein-tyrosine phosphatases, PTP1 and YOP. The ability to observe distinct phases of the reaction mechanism during OMFP hydrolysis will facilitate future analysis of critical catalytic residues in Cdc25 and other dual-specificity phosphatases.

Protein and ligand dynamics in 4-hydroxybenzoate hydroxylase.

para-Hydroxybenzoate hydroxylase catalyzes a two-step reaction that demands precise control of solvent access to the catalytic site. The first step of the reaction, reduction of flavin by NADPH, requires access to solvent. The second step, oxygenation of reduced flavin to a flavin C4a-hydroperoxide that transfers the hydroxyl group to the substrate, requires that solvent be excluded to prevent breakdown of the hydroperoxide to oxidized flavin and hydrogen peroxide. These conflicting requirements are met by the coordination of multiple movements involving the protein, the two cofactors, and the substrate. Here, using the R220Q mutant form of para-hydroxybenzoate hydroxylase, we show that in the absence of substrate, the large βαβ domain (residues 1–180) and the smaller sheet domain (residues 180–270) separate slightly, and the flavin swings out to a more exposed position to open an aqueous channel from the solvent to the protein interior. Substrate entry occurs by first binding at a surface site and then sliding into the protein interior. In our study of this mutant, the structure of the complex with pyridine nucleotide was obtained. This cofactor binds in an extended conformation at the enzyme surface in a groove that crosses the binding site of FAD. We postulate that for stereospecific reduction, the flavin swings to an out position and NADPH assumes a folded conformation that brings its nicotinamide moiety into close contact with the isoalloxazine moiety of the flavin. This work clearly shows how complex dynamics can play a central role in catalysis by enzymes.

Reactivity of thioredoxin as a protein thiol-disulfide oxidoreductase.

Thiol-disulfide reactions are crucial for redox homeostasis in the cell.1-2 As a disulfide oxidoreductase, thioredoxin (Trx, ∼12 kDa) regulates a wide variety of biological molecules in both eukaryotic and prokaryotic species, including ribonucleotide reductase, peroxiredoxin, methionine sulfoxide reductase, phosphatase and tensin homolog (PTEN), transcription factors such as nuclear factor-κB (NF-κB), redox factor-1 (Ref-1), and activator protein-1 (AP-1).3 Thus, Trx has been implicated in such diverse processes as antioxidant defense, DNA repair and synthesis, redox regulation, and apoptosis.4-9 In the evolution of Trx catalysis important physical factors (e.g., a hydrophobic binding groove) have appeared to make Trx bind the disulfide substrate in a specific fashion and generate stabilizing interactions.10-11 The interactions and substrate binding were shown to regulate the geometry and orientation of the target disulfide in the catalytic site of the enzyme, and account for Michaelis-Menten–type kinetics of disulfide reduction by Trx.10-11 Trx molecules contain two cysteines in the active site in a CXXC motif. The chemistry of Trx-catalyzed protein disulfide reduction following substrate-binding includes a nucleophilic attack on the disulfide of target proteins by the N-terminal cysteine thiolate of Trx (which is Cys32 in the E. coli numbering) to form an intermolecular mixed disulfide, and a subsequent attack on the disulfide intermediate by the thiolate of the C-terminal cysteine of Trx (Cys35), producing the reduced target protein and oxidized Trx (Figure 1).12-13 To date a number of factors, including the identity of the amino acid residues spanning the CXXC motif, the pKa of the redox active thiols, the redox potential of the disulfide/dithiol couple, the acid/base catalysts, the molecular interactions and the local conformational changes after substrate binding, have been implicated in the regulation of Trx activity as a protein disulfide oxidoreductase, albeit with controversies and debates. In this work, we review the emerging evidence that support or dispute the regulating roles of these factors, and propose the likely prime determinants of Trx activity that deserve more attention in future research, with the hope to promote a better understanding of the mechanistic factors of Trx reactivity. Figure 1 A schematic cartoon view of a Trx-catalyzed disulfide reduction. A number of factors are involved in the regulation of Trx activity, including the amino acid residues spanning the redox active CXXC motif, molecular interaction (e.g., electrostatic force), ... The 3-D structures of Trx proteins are highly conserved, with the five central β-strands surrounded by four α-helices (Figure 2, A and B). Part of the redox active center (-CGPC- motif) protrudes at the surface of the molecule at the very N-terminus of helix α2, with Cys35 largely covered by the N-terminal portion of the helix α2. Only one side of the Cys32-SH is accessible to solvent for the transfer of reducing equivalents in Trxox but the side chain of Cys32 turns into the solvent upon reduction.14-16 The Trx fold is also found in several other classes of enzymes that interact with substrates containing either disulfides or dithiols. Protein families that have a Trx-fold include the thioredoxin, Dsb (disulfide bond formation protein) proteins, glutaredoxin (Grx), glutathione S-transferase, and protein disulfide isomerase (PDI) families.2,16-17 We performed a structural bioinformatics study on 515 sequences of the Trx family, 495 sequences of the DsbA family, and 382 sequences of the PDI family (from the Conserved Domain Database (CDD),18 http://www.ncbi.nlm.nih.gov/sites/entrez?db=cdd). This search confirmed a recent observation that the two components that are most conserved across these 3 families of proteins include an active site CXXC motif, and a conserved proline that is distant in sequence from this active site but immediately adjacent in 3 dimensional space.19 In the Trx family, the CXXC motif has the sequence CGPC and is present at position 32–35, while the proline residue is at position 76 in its best-studied member, E. coli thioredoxin 1 (EcTrx; PDB: 2TRX_A) (Figure 2C). The highly conserved proline is at position 151 in E. coli DsbA (PDB: 1DSB_A) and at position 83 in human PDI (PDB: 1MEK) (Figure 2, D and E). In EcTrx, the distances between the N-atom of Pro76 and the two sulfur atoms of the cysteine pair are 4.07 A (Cys32) and 3.62A (Cys35) (Figure 2F). The next closest residue is Asp26, and the shortest distance between an O-atom of Asp26 and the sulfur atom of Cys35 is 5.59 A (Figure 2G). Pro76 and Asp26 have been shown to play important roles in the redox reactions due to their close contact with the Cys pair.20-21 Figure 2 The structure of EcTrx and conserved amino acid residues in the Trx family. (A) and (B) are the ribbon diagrams of oxidized and reduced EcTrx, respectively. They show the spatial distances between Trp28 and Cys32, and between Trp28 and Asp26. The color ... Conserved structural features such as the Trx-fold and secondary structure in Trx family members, never-the-less, allow diverse reactivities in catalyzing protein disulfide interchange reactions. Quantum mechanical calculations suggest that the relative stability of thiolates in the CXXC motif determines whether these enzymes catalyze oxidation, reduction, or isomerization.2,22-23 Because the cysteine thiolates in Trx are both poorly stabilized, Trx is a good reducing agent. In contrast, DsbA stabilizes both cysteine thiolates and thus is a good oxidizing agent; isomerases such as PDI have one thiolate relatively solvent exposed and poorly stabilized while the other (relatively buried) thiolate is highly stabilized.22-23 Static calculations, such as the quantum mechanical calculations, provide insights into the thermodynamics of thiol-disulfide reactions, but as previously noted and as recently further investigated,1,24 the reactivity in an actual reaction is more complex due to dynamics and conformational changes that occur during substrate binding. For instance, studies with site-specific mutagenesis indicate that the relative stability of the oxidized versus the reduced form determines the difference in the redox potentials of Trx from Staphylococcus aureus (SaTrx).25-27 Replacement of the conserved proline in the CXXC motif by threonine or serine greatly reduced the relative stability value and made SaTrx less reducing.25 Moreover, the activation energy barrier for forming a transition state complex must be overcome by dynamics or other properties to accomplish a thermodynamically favorable thiol-disulfide reaction.1 However, DsbA from Staphylococcus aureus shows identical stabilities in oxidized and reduced forms, suggesting that alternative mechanisms beyond thermodynamic stability underlie the activity of SaDsbA in thiol-disulfide reactions.28-29 In this work, we review recent studies on the reactivity of thioredoxins as a protein disulfide oxidoreductase. Our goal is to provide an updated view and a better understanding of the determinants of Trx reactivity and their critical roles in redox homeostasis.

Kinetic Mechanisms of the Oxygenase from a Two-component Enzyme, p-Hydroxyphenylacetate 3-Hydroxylase from Acinetobacter baumannii *

p-Hydroxyphenylacetate hydroxylase (HPAH) from Acinetobacter baumannii catalyzes the hydroxylation of p-hydroxyphenylacetate (HPA) to form 3,4-dihydroxyphenylacetate (DHPA). The enzyme system is composed of two proteins: an FMN reductase (C1) and an oxygenase that uses FMNH– (C2). We report detailed transient kinetics studies at 4 °C of the reaction mechanism of C2.C2 binds rapidly and tightly to reduced FMN (Kd, 1.2 ± 0.2 μm), but less tightly to oxidized FMN (Kd, 250 ± 50 μm). The complex of C -FMNH–2 reacted with oxygen to form C(4a)-hydroperoxy-FMN at 1.1 ± 0.1 × 106 m–1 s–1, whereas the C -FMNH–2 -HPA complex reacted with oxygen to form C(4a)-hydroperoxy-FMN-HPA more slowly (k = 4.8 ± 0.2 × 104 m–1 s–1). The kinetic mechanism of C2 was shown to be a preferential random order type, in which HPA or oxygen can initially bind to the C -FMNH–2 complex, but the preferred path was oxygen reacting with C -FMNH–2 to form the C(4a)-hydroperoxy-FMN intermediate prior to HPA binding. Hydroxylation occurs from the ternary complex with a rate constant of 20 s–1 to form the C2-C(4a)-hydroxy-FMN-DHPA complex. At high HPA concentrations (>0.5 mm), HPA formed a dead end complex with the C2-C(4a)-hydroxy-FMN intermediate (similar to single component flavoprotein hydroxylases), thus inhibiting the bound flavin from returning to the oxidized form. When FADH– was used, C(4a)-hydroperoxy-FAD, C(4a)-hydroxy-FAD, and product were formed at rates similar to those with FMNH–. Thus, C2 has the unusual ability to use both common flavin cofactors in catalysis.

Structure and mechanism of the iron-sulfur flavoprotein phthalate dioxygenase reductase.

Transfer of electrons between pyri‐dine nucleotides (obligatory two‐electron carriers) and hemes or [2Fe‐2S] centers (obligatory one‐electron carriers) is an essential step mediated by flavins in respiration, photosynthesis, and many oxygenase systems. Phthalate dioxygenase reductase (PDR), a soluble iron‐sulfur flavoprotein from Pscudomofias cepacia, is a convenient model for the study of this type of electron transfer. PDR is folded into thrjee domains; the NH2terminal FMN binding and central NAD(H) binding domains are closely related to ferredoxin‐NADP+ reductase (FNR). The COOH‐términal [2Fe‐2S] domain is similar to plaUt ferredoxins, and can be removed by proteolysis without significantly altering the reactivity of the FNR‐like domains. Kinetic studies have identified sequential steps in the reaction of PDR with NADH that involve pyridine nucleotide binding, hydrijde transfer to FMN, and intramolecular electron transfer from the reduced flavin to the [2Fe‐2S] cluster. Crystal structures of reduced and liganded PDR correspond to some of the intermediates formed during reduction by NADH. Small structural changes that are observed in the vicinity of the cofactors upon reduction or NAD(H) binding may provide part of the reorganization energy or contribute to the gating mechanism that controls intramolecular electron transfer.—Gassner, G. T., Ludwig, M. L., Gatti, D. L., Correll, C. C., Ballou, D. P. Structure and mechanism of the iron‐sulfur flavoprotein phthalate dioxygenase reductase. FASEB J. 9, 1411‐1418 (1995)