title: Deciphering structural and functional roles of individual disulfide bonds of the mitochondrial sulfhydryl oxidase Erv1p.
abstract: Erv1p is a FAD-dependent sulfhydryl oxidase of the mitochondrial intermembrane space. It contains three conserved disulfide bonds arranged in two CXXC motifs and one CX(16)C motif. Experimental evidence for the specific roles of the individual disulfide bonds is lacking. In this study, structural and functional roles of the disulfides were dissected systematically using a wide range of biochemical and biophysical methods. Three double cysteine mutants with each pair of cysteines mutated to serines were generated. All of the mutants were purified with the normal FAD binding properties as the wild type Erv1p, showing that none of the three disulfides are essential for FAD binding. Thermal denaturation and trypsin digestion studies showed that the CX(16)C disulfide plays an important role in stabilizing the folding of Erv1p. To understand the functional role of each disulfide, small molecules and the physiological substrate protein Mia40 were used as electron donors in oxygen consumption assays. We show that both CXXC disulfides are required for Erv1 oxidase activity. The active site disulfide is well protected thus requires the shuttle disulfide for its function. Although both mutants of the CXXC motifs were individually inactive, Erv1p activity was partially recovered by mixing these two mutants together, and the recovery was rapid. Thus, we provided the first experimental evidence of electron transfer between the shuttle and active site disulfides of Erv1p, and we propose that both intersubunit and intermolecular electron transfer can occur.
authors.affiliation: Faculty of Life Sciences, The University of Manchester, Michael Smith Building, Oxford Road, Manchester M13 9PT, United Kingdom.
authors.forename: Swee Kim
authors.initials: SK
authors.lastname: Ang
authors.forename: Hui
authors.initials: H
authors.lastname: Lu
mesh_terms: D020816:Amino Acid Motifs; D020134:Catalytic Domain; D002942:Circular Dichroism; D004220:Disulfides / Q000737:chemistry; D004583:Electrons; D007700:Kinetics; D008928:Mitochondria / Q000201:enzymology*; D024101:Mitochondrial Proteins / Q000737:chemistry* / Q000502:physiology*; D008957:Models, Genetic; D008968:Molecular Conformation; D009154:Mutation; D050862:Oxidoreductases Acting on Sulfur Group Donors / Q000737:chemistry* / Q000502:physiology*; D010101:Oxygen Consumption; D011487:Protein Conformation; D017434:Protein Structure, Tertiary; D012441:Saccharomyces cerevisiae / Q000378:metabolism; D029701:Saccharomyces cerevisiae Proteins / Q000737:chemistry* / Q000502:physiology*
chemical_list: D004220:Disulfides; D024101:Mitochondrial Proteins; D029701:Saccharomyces cerevisiae Proteins; D050862:Oxidoreductases Acting on Sulfur Group Donors; C073790:ERV1 protein, S cerevisiae
references.citation: J Biol Chem. 2007 May 11;282(19):13929-33
references.pmid: 17353193
references.citation: J Biol Chem. 2009 Mar 13;284(11):6818-25
references.pmid: 19117943
references.citation: Eur J Biochem. 2003 Apr;270(7):1528-35
references.pmid: 12654008
references.citation: Biochim Biophys Acta. 2009 Jan;1793(1):139-45
references.pmid: 18761382
references.citation: Cell. 2005 Jul 1;121(7):965-7
references.pmid: 15989945
references.citation: Antioxid Redox Signal. 2006 May-Jun;8(5-6):797-811
references.pmid: 16771671
references.citation: J Mol Biol. 2005 Nov 11;353(5):937-44
references.pmid: 16185707
references.citation: Proc Natl Acad Sci U S A. 1992 Sep 1;89(17):7944-8
references.pmid: 1518818
references.citation: Biochim Biophys Acta. 2008 Apr;1783(4):557-66
references.pmid: 18155671
references.citation: EMBO J. 2008 Nov 19;27(22):2988-97
references.pmid: 18971943
references.citation: EMBO J. 2008 Nov 19;27(22):2977-87
references.pmid: 18833192
references.citation: Biochim Biophys Acta. 2008 Apr;1783(4):601-9
references.pmid: 18179776
references.citation: FEBS Lett. 2000 Jul 14;477(1-2):62-6
references.pmid: 10899311
references.citation: Cell. 2004 May 28;117(5):601-10
references.pmid: 15163408
references.citation: Cell. 2007 Apr 20;129(2):333-44
references.pmid: 17448992
references.citation: Nat Struct Mol Biol. 2009 Feb;16(2):198-206
references.pmid: 19182799
references.citation: Annu Rev Biochem. 2003;72:111-35
references.pmid: 12524212
references.citation: Cell. 2005 Jul 1;121(7):1059-69
references.pmid: 15989955
references.citation: Antioxid Redox Signal. 2006 Mar-Apr;8(3-4):300-11
references.pmid: 16677076
references.citation: J Mol Biol. 2006 Sep 8;362(1):89-101
references.pmid: 16893552
references.citation: J Biol Chem. 2004 Apr 30;279(18):18952-8
references.pmid: 14973127
references.citation: Inorg Chem. 2003 Mar 24;42(6):1994-2003
references.pmid: 12639134
references.citation: J Biol Chem. 2009 Jan 2;284(1):199-206
references.pmid: 19001419
references.citation: Trends Cell Biol. 2000 May;10(5):203-10
references.pmid: 10754564
references.citation: Mol Microbiol. 2002 Apr;44(1):1-8
references.pmid: 11967064
references.citation: EMBO J. 2007 Nov 28;26(23):4801-11
references.pmid: 17972915
references.citation: J Biol Chem. 2008 Oct 24;283(43):29126-34
references.pmid: 18708636
references.citation: J Mol Biol. 2005 Nov 4;353(4):897-910
references.pmid: 16199054
references.citation: J Cell Biol. 2008 Oct 20;183(2):195-202
references.pmid: 18852299
references.citation: Proc Natl Acad Sci U S A. 2006 Jan 10;103(2):299-304
references.pmid: 16407158
references.citation: J Biol Chem. 2007 Dec 28;282(52):37461-70
references.pmid: 17959605
references.citation: Biochim Biophys Acta. 2008 Apr;1783(4):610-7
references.pmid: 17996737
references.citation: Mol Biol Cell. 2008 Jan;19(1):226-36
references.pmid: 17978092
references.citation: Nat Struct Biol. 2002 Jan;9(1):61-7
references.pmid: 11740506
references.citation: Mol Biol Cell. 2006 May;17(5):2256-66
references.pmid: 16495342