Colin Thorpe

An acyl-coenzyme a dehydrogenase assay utilizing the ferricenium ion

A sensitive assay for medium chain acyl-CoA dehydrogenase has been developed by substituting ferricenium hexafluorophosphate for the physiological acceptor, electron transferring flavoprotein. The ferricenium ion is a facile oxidant of the octanoyl-CoA-reduced enzyme with a Vmax of 1400 min-1 and a KM of 55 microM at pH 7.6. The ferricenium assay does not require additional mediator dyes, exhibits low background rates, and avoids the necessity of purifying substantial amounts of electron transferring flavoprotein. Unlike the fluorescence-based electron transferring flavoprotein assay, this new procedure can be performed aerobically. Both assays give comparable results when tested with crude fibroblast homogenates from normal and medium chain acyl-CoA dehydrogenase deficient patients. The convenience of the ferricenium method suggests it may be generally useful as a screening assay for a number of acyl-CoA dehydrogenases.

Sulfhydryl oxidases: emerging catalysts of protein disulfide bond formation in eukaryotes.

Members of the Quiescin-sulfhydryl oxidase (QSOX) family utilize a thioredoxin domain and a small FAD-binding domain homologous to the yeast ERV1p protein to oxidize sulfhydryl groups to disulfides with the reduction of oxygen to hydrogen peroxide. QSOX enzymes are found in all multicellular organisms for which complete genomes exist and in Trypanosoma brucei, but are not found in yeast. The avian QSOX is the best understood enzymatically: its preferred substrates are peptides and proteins, not monothiols such as glutathione. Mixtures of avian QSOX and protein disulfide isomerase catalyze the rapid insertion of the correct disulfide pairings in reduced RNase. Immunohistochemical studies of human tissues show a marked and highly localized concentration of QSOX in cell types associated with heavy secretory loads. Consistent with this role in the formation of disulfide bonds, QSOX is typically found in the cell in the endoplasmic reticulum and Golgi and outside the cell. In sum, this review suggests that QSOX enzymes play a significant role in oxidative folding of a large variety of proteins in a wide range of multicellular organisms.

Structure and mechanism of action of the acyl-CoA dehydrogenases.

Mitochondrial β‐oxidation involves a family of flavoproteins that introduce a C‐C double bond into their fatty acyl‐CoA substrates. Deficiencies of these acyl‐CoA dehydrogenases lead to fatty acid oxidation disorders involving life‐threatening episodes of metabolic derangement. This review focuses on the medium chain acyl‐CoA dehydrogenase as the best‐understood member of its class. The crystal structure of the enzyme and salient features of its substrate specificity and mechanism of action are summarized. The surprising observation of a catalyti‐cally essential amino acid residue that nevertheless is not conserved in the acyl‐CoA dehydrogenase family is discussed.—Thorpe, C., Kim, J‐J. P. Structure and mechanism of action of the acyl‐CoA dehydrogenases. FASEB J. 9, 718‐725 (1995)

Sulfhydryl Oxidase from Egg White A FACILE CATALYST FOR DISULFIDE BOND FORMATION IN PROTEINS AND PEPTIDES

Both metalloprotein and flavin-linked sulfhydryl oxidases catalyze the oxidation of thiols to disulfides with the reduction of oxygen to hydrogen peroxide. Despite earlier suggestions for a role in protein disulfide bond formation, these enzymes have received comparatively little general attention. Chicken egg white sulfhydryl oxidase utilizes an internal redox-active cystine bridge and a FAD moiety in the oxidation of a range of small molecular weight thiols such as glutathione, cysteine, and dithiothreitol. The oxidase is shown here to exhibit a high catalytic activity toward a range of reduced peptides and proteins including insulin A and B chains, lysozyme, ovalbumin, riboflavin-binding protein, and RNase. Catalytic efficiencies are up to 100-fold higher than for reduced glutathione, with typical K m values of about 110–330 μm/protein thiol, compared with 20 mm for glutathione. RNase activity is not significantly recovered when the cysteine residues are rapidly oxidized by sulfhydryl oxidase, but activity is efficiently restored when protein disulfide isomerase is also present. Sulfhydryl oxidase can also oxidize reduced protein disulfide isomerase directly. These data show that sulfhydryl oxidase and protein disulfide isomerase can cooperate in vitro in the generation and rearrangement of native disulfide pairings. A possible role for the oxidase in the protein secretory pathway in vivo is discussed.

A Sulfhydryl Oxidase from Chicken Egg White

A dimeric glycoprotein containing one FAD per ∼80,000 Mr subunit has been isolated from chicken egg white and found to have sulfhydryl oxidase activity with a range of small molecular weight thiols. Dithiothreitol was the best substrate of those tested, with a turnover number of 1030/min, a Km of 150 μM, and a pH optimum of about 7.5. Oxidation of thiol substrates generates hydrogen peroxide in aerobic solution. Anaerobically, the ferricenium ion is a facile alternative electron acceptor. Reduction of the oxidase with dithionite or dithiothreitol under anaerobic conditions yields a two-electron intermediate (EH2) showing a charge transfer band (λmax 560 nm; εobs 2.5 mM−1 cm−1). Complete bleaching of the flavin and discharge of the charge transfer complex require a total of four electrons. Borohydride and catalytic photoreduction give the same spectral changes. EH2, but not the oxidized enzyme, is inactivated by iodoacetamide with alkylation of 2.7 cysteine residues/subunit. These data indicate that the oxidase contains a redox-active disulfide bridge generating a thiolate to oxidized flavin charge transfer complex at the EH2 level. Sulfite treatment does not form the expected flavin adduct with the native enzyme but cleaves the active site disulfide, yielding an air-stable EH2-like species. The close functional resemblance of the oxidase to the pyridine nucleotide-dependent disulfide oxidoreductase family is discussed.

Quantification of Thiols and Disulfides

BACKGROUND Disulfide bond formation is a key posttranslational modification, with implications for structure, function and stability of numerous proteins. While disulfide bond formation is a necessary and essential process for many proteins, it is deleterious and disruptive for others. Cells go to great lengths to regulate thiol-disulfide bond homeostasis, typically with several, apparently redundant, systems working in parallel. Dissecting the extent of oxidation and reduction of disulfides is an ongoing challenge due, in part, to the facility of thiol/disulfide exchange reactions. SCOPE OF REVIEW In the present account, we briefly survey the toolbox available to the experimentalist for the chemical determination of thiols and disulfides. We have chosen to focus on the key chemical aspects of current methodology, together with identifying potential difficulties inherent in their experimental implementation. MAJOR CONCLUSIONS While many reagents have been described for the measurement and manipulation of the redox status of thiols and disulfides, a number of these methods remain underutilized. The ability to effectively quantify changes in redox conditions in living cells presents a continuing challenge. GENERAL SIGNIFICANCE Many unresolved questions in the metabolic interconversion of thiols and disulfides remain. For example, while pool sizes of redox pairs and their intracellular distribution are being uncovered, very little is known about the flux in thiol-disulfide exchange pathways. New tools are needed to address this important aspect of cellular metabolism. This article is part of a Special Issue entitled Current methods to study reactive oxygen species - pros and cons and biophysics of membrane proteins. Guest Editor: Christine Winterbourn.

Oxidative Protein Folding and the Quiescin–Sulfhydryl Oxidase Family of Flavoproteins

Flavin-linked sulfhydryl oxidases participate in the net generation of disulfide bonds during oxidative protein folding in the endoplasmic reticulum. Members of the Quiescin-sulfhydryl oxidase (QSOX) family catalyze the facile direct introduction of disulfide bonds into unfolded reduced proteins with the reduction of molecular oxygen to generate hydrogen peroxide. Current progress in dissecting the mechanism of QSOX enzymes is reviewed, with emphasis on the CxxC motifs in the thioredoxin and Erv/ALR domains and the involvement of the flavin prosthetic group. The tissue distribution and intra- and extracellular location of QSOX enzymes are discussed, and suggestions for the physiological role of these enzymes are presented. The review compares the substrate specificity and catalytic efficiency of the QSOX enzymes with members of the Ero1 family of flavin-dependent sulfhydryl oxidases: enzymes believed to play key roles in disulfide generation in yeast and higher eukaryotes. Finally, limitations of our current understanding of disulfide generation in metazoans are identified and questions posed for the future.

Homology between Egg White Sulfhydryl Oxidase and Quiescin Q6 Defines a New Class of Flavin-linked Sulfhydryl Oxidases

The flavin-dependent sulfhydryl oxidase from chicken egg white catalyzes the oxidation of sulfhydryl groups to disulfides with the reduction of oxygen to hydrogen peroxide. Reduced proteins are the preferred thiol substrates of this secreted enzyme. The egg white oxidase shows an average 64% identity (from randomly distributed peptides comprising more than 30% of the protein sequence) to a human protein, Quiescin Q6, involved in growth regulation. Q6 is strongly expressed when fibroblasts enter reversible quiescence (Coppock, D. L., Cina-Poppe, D., Gilleran, S. (1998)Genomics 54, 460–468). A peptide antibody against Q6 cross-reacts with both the egg white enzyme and a flavin-linked sulfhydryl oxidase isolated from bovine semen. Sequence analyses show that the egg white oxidase joins human Q6, bone-derived growth factor, GEC-3 from guinea pig, and homologs found in a range of multicellular organisms as a member of a new protein family. These proteins are formed from the fusion of thioredoxin and ERV motifs. In contrast, the flavin-linked sulfhydryl oxidase from Aspergillus niger is related to the pyridine nucleotide-dependent disulfide oxidoreductases, and shows no detectable sequence similarity to this newly recognized protein family.

Generating Disulfides in Multicellular Organisms: Emerging Roles for a New Flavoprotein Family

Peptides and proteins destined for secretion in multicellular organisms usually contain disulfide bonds, from small peptides tomassive extracellular matrix (ECM)2 proteins with hundreds of disulfide bridges. Disulfides are important to the structure, stability, and regulation of many proteins having at least one extracellular domain; they are critical to the formation and remodeling of the ECM and other disulfide networks, and they are crucial elements in various redox signaling pathways. However, the pathways for their biosynthesis in multicellular organisms remain surprisingly cryptic.We do not really knowhow a single protein disulfide bond is introduced in any metazoan, green plant, or protist. Why is our understanding of oxidative folding in so rudimentary a state? One reason is the very reactivity of thiolate nucleophiles and the degeneracy of pathways for the interconversion of thiols and disulfides. A second factor is the facile non-enzymatic oxidation of thiols by a number of potential cellular oxidants including GSSG (1). A third issue is the commonmisperception that oxygen is a facile oxidant of juxtaposed thiols, a reaction that is spin-forbidden and strongly catalyzed by traces of redox-active transitionmetal ions (notably copper and iron). Finally, multicellular organisms have additional pathways for disulfide bond formation that are not shared with the genetically tractable yeast systems.

Methodology employed for anaerobic spectrophotometric titrations and for computer-assisted data analysis.

Publisher Summary This chapter describes the methodology employed for anaerobic spectrophotometric titrations and for computer-assisted data analysis. Many proteins that catalyze oxidoreductions have chromophoric prosthetic groups that permit the state of reduction of the protein to be monitored spectrophotometrically. The comparison of spectral data generated in the chemical manipulations of flavoproteins (or any solution with visible or ultraviolet absorbance) has been facilitated by interfacing a recording spectrophotometer with a minicomputer. Spectra can be stored on magnetic tape in a standard format and can be recalled for comparisons, mathematical manipulations, or hard-copy output. Spectra are visualized on the display terminal for comparison or manipulation, and hard-copy is made on the X-Y recorder. The procedures used for preparation of the anaerobic solutions for titration and the subsequent analysis of the data are illustrated in the chapter by describing in detail the protocol for the measurement of proton release associated with two electron reduction of lipoamide dehydrogenase by dihydrolipoamide. In these experiments, the enzyme is dissolved in an unbuffered solution containing phenol red as a pH indicator and is then reduced with dihydrolipoamide under anaerobic conditions.