Supplementary teaching slides for: Generating disulfides in multicellular organisms: emerging roles for a new flavoprotein family Colin Thorpe and Donald L. Coppock November 2006 ct 1

Phospholipase A2 Insulin Laminin, four domains Disulfides bonds are frequently found in secreted eukaryotic proteins – examples … RNAse ct 2

Different cofactors support oxidative protein folding in prokaryotes and eukaryotes Electron transport chain SH SH DsbA S S SS O 2 fumarate S S Q DsbB Escherichia coli periplasm: coenzyme Q (bound to DsbB) connects thiol oxidation with the respiratory chain. Note series of redox-active disulfides. Arrows represent flow of electron pairs. In yeast, two flavoproteins, Ero1p and Erv2p, can cooperate with PDI (flavin ) Yeast endoplasmic reticulum: flavin involved in at least two alternate pathways. cytosol periplasm SH SH Oxygen PDI Ero1p Erv2p ct 3

Multicellular organisms have additional options for inserting disulfide bonds In humans: Ero1 and QSOX (Erv2p is absent). Ero enzymes receive pairs of electrons from client proteins undergoing oxidative protein folding. PDI serves as a mediator. QSOX enzymes insert disulfides directly into the client proteins. PDI functions later. PDI acts both as a disulfide oxidoreductase and a disulfide isomerase (shuffling incorrect –S-S- bonds in a redox-neutral way). QSOX SH SH Oxygen PDI Ero1 QSOX PDI Other pathways for oxidative protein folding? Next: generating disulfides using flavin and oxygen ct 4

In eukaryotes, flavoenzymes are frequently used to generate disulfides. The net oxidant is molecular oxygen: Flavin-dependent sulfhydryl oxidases catalyze this reaction in a series of steps: sulfhydryl oxidase ct 5

The first crystal structure of a sulfhydryl oxidase was of yeast Erv2p (Gross et al., 2002) The numbered steps are shown above and to the left 1. Input of 2-electrons from substrate by disulfide exchange. 2.A series of internal disulfide exchanges (here just one) leads to reduction of the disulfide proximal to the flavin (FAD). 3.Two-electron reduction of the flavin. 4.Reoxidation of reduced flavin by molecular oxygen generating H 2 O 2 and reforming oxidized enzyme. Key stages of catalysis in a typical flavin-dependent sulfhydryl oxidase: sulfhydryl oxidase ct 6

Step 1: the initial reduction of the enzyme by the substrate dithiol. Flow of reducing equivalents = Donor dithiol (substrate) Acceptor disulfide (on oxidase) Key stages of sulfhydryl oxidase catalysis: steps 1 and 2 (multiple disulfide exchange steps are common) 2e - sulfhydryl oxidase 2e - ct 7

Key stages of sulfhydryl oxidase catalysis: the flavin connects oxidation of thiols with the reduction of O 2 N N NH N O O N H N NH N O O S SH S SH(S - ) N H N NH N O O S S 2e - sulfhydryl oxidase Step 3, reduction of the flavin prosthetic group (a transient cysteinyl-adduct forms with the flavin) ct 8

Key stages of sulfhydryl oxidase catalysis: step 4, reoxidation of the flavin prosthetic group Step 4, reoxidation of the flavin prosthetic group by oxygen (reaction proceeds via a diradical pair : not shown) Key stages of sulfhydryl oxidase catalysis: reoxidation of the enzyme and the generation of H 2 O 2 2e - sulfhydryl oxidase ct 9

Structures of yeast Erv2p and Ero1p. Both use a series of disulfide exchanges and a flavin cofactor S H S H P D I Ero1p 1 4 oxygen S H S H P D I Erv2p ct 10

QSOX - an ancient fusion of recognizable domains … metazoan QSOX depicted here Thioredoxin domains Trx1 and 2 Resembling the first two thioredoxin domains in PDI ERV/ALR domain An Erv homolog is called ALR (augmenter of liver regeneration) -WCGHC- ct 11

QSOX enzymes have 1 or 2 thioredoxin domains. Differential splicing at C-terminus yields a short form Mechanism: thioredoxin domain reacts with substrate dithiols. Steps 2-5 are by analogy with Erv2p -CxxC--CxxC--CxxC- O HS HS PDI - like ct 12

QSOX oxidizes reduced protein substrate, PDI isomerizes incorrectly placed disulfides ct 131

Unresolved general questions for disulfide bond formation in multicellular organisms The relative importance of the pathways depicted here. The precise roles of Ero1  and Ero1  and QSOX1 and QSOX2 … distinct or overlapping substrate specificities. How hydrogen peroxide, generated by these oxidases, is handled in the ER. How the redox state of PDI is maintained in the ER to optimize formation of correctly-paired disulfide bonds in client proteins. SH SH Oxygen PDI Ero1 QSOX PDI Other pathways for oxidative protein folding? ct 14