Supplementary teaching slides for:

Generating disulfides in multicellular organisms: emerging roles for a new flavoprotein family

Colin Thorpe and Donald L. Coppock

November 2006

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Phospholipase A2

Insulin

Laminin, four domains

Disulfides bonds are frequently found in secreted eukaryotic proteins – examples …

RNAse

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Different cofactors support oxidative protein folding in prokaryotes and eukaryotes

Electron transport chain

SHSH

DsbA

SSS S

O2fumarate

S S

Q

DsbB

Escherichia coliperiplasm: 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

SHSH OxygenPDI

Ero1p

Erv2p

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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).

Q S O X

SHSH Oxygen

PDI Ero1

QSOXPDI

Other pathways for oxidative protein folding?

Next: generating disulfides using flavin and oxygen

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In eukaryotes, flavoenzymes are frequently used to generate disulfides. The net oxidant is molecular oxygen:

SH

SH

SS

S

S

HS

HS S

S

SH

SHFlox

Flred O2

H2O2

1 2 3 4

Flavin-dependent sulfhydryl oxidases catalyze this reaction in a series of steps:

NH

HN

SO

O

HN

NH

SO

OHN

NH

HSO

O

NH

HN

SHO

O

O2 H2O2+ +enzyme

sulfhydryl oxidase

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oxygen

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 H2O2

and reforming oxidized enzyme.

Key stages of catalysis in a typical flavin-dependent sulfhydryl oxidase:

SH

SH

SS

S

S

HS

HS S

S

SH

SHFlox

Flred O2

H2O2

1 2 3 4

sulfhydryl oxidase

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Step 1: the initial reduction of the enzyme by the substrate dithiol. Flow of reducing equivalents =

SH

S

SS

S

SBH

SH

SS

HS

B

S

HS

BH

Donor dithiol(substrate)

Acceptor disulfide(on oxidase)

Key stages of sulfhydryl oxidase catalysis: steps 1 and 2 (multiple disulfide exchange steps are common)

2e-

SH

SH

SS

S

S

HS

HS S

S

SH

SHFlox

Flred O2

H2O2

1 2 3 4

sulfhydryl oxidase

2e-

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Key stages of sulfhydryl oxidase catalysis: the flavin connects oxidation of thiols with the reduction of O2

N

N

NH

N O

ONH

N

NH

N O

O

S

SH

S

SH(S-)

NH

N

NH

N O

O

S

S

2e-

SH

SH

SS

S

S

HS

HS S

S

SH

SHFlox

Flred O2

H2O2

1 2 3 4

sulfhydryl oxidase

Step 3, reduction of the flavin prosthetic group (a transient cysteinyl-adduct forms with the flavin)

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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)

NH

N

NH

N O

O

NH

N

NH

N O

OON

N

NH

N O

O

O

O

OH

H+

H+

HOOH

Key stages of sulfhydryl oxidase catalysis: reoxidation of the enzyme and the generation of H2O2

2e-

SH

SH

SS

S

S

HS

HS S

S

SH

SHFlox

Flred O2

H2O2

1 2 3 4

sulfhydryl oxidase

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Structures of yeast Erv2p and Ero1p. Both use a series of disulfide exchanges and a flavin cofactor

SH

SHPDI

Ero1p

1

4

oxygen

SH

SHPDI

E rv 2 p

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Signal Trx1 Trx2 Spacer ERV/ALR Transmembrane

N CCxxC CxxCCxxC

FAD

QSOX - an ancient fusion of recognizable domains … metazoan QSOX depicted here

Thioredoxin domains Trx1 and 2Resembling the first two thioredoxin domains in PDI

ERV/ALR domainAn Erv homolog is called ALR (augmenter of liver regeneration)

-WCGHC-

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Human QSOX1

Signal Trx1 Trx2 Spacer Erv/ALR Transmembrane

N CCxxC CxxCCxxC

CxxC CxxCCxxC

CxxC CxxCCxxC

C

C

N

N

FAD

FAD

FAD

Arabidopsis QSOX1

Trypanosoma brucei QSOX

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-

O2

1

2 3

4

5

H SH S

PDI - like

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QSOX oxidizes reduced protein substrate, PDI isomerizes incorrectly placed disulfides

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Unresolved general questions for disulfide bond formation in multicellular organisms

The relative importance of the pathways depicted here.

The precise roles of Ero1and 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.

SHSH Oxygen

PDI Ero1

QSOXPDI

Other pathways for oxidative protein folding?

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