Reconstitution of mitochondrial processing peptidase from the core proteins (subunits I and II) of bovine heart mitochondrial cytochrome bc(1) complex.
Mature core I and core II proteins of the bovine heart mitochondrial cytochrome bc(1) complex were individually overexpressed in Escherichia coli as soluble proteins using the expression vector pET-I and pET-II, respectively. Purified recombinant core I and core II alone show no mitochondrial processing peptidase (MPP) activity. When these two proteins are mixed together, MPP activity is observed. Maximum activity is obtained when the molar ratio of these two core proteins reaches 1. This indicates that only the two core subunits of thebc(1) complex are needed for MPP activity. The properties of reconstituted MPP are similar to those of Triton X-100-activated MPP in the bovine bc(1) complex. When Rieske iron-sulfur protein precursor is used as substrate for reconstituted MPP, the processing activity stops when the amount of product formation (subunit IX) equals the amount of reconstituted MPP used in the system. Addition of Triton X-100 to the product-inhibited reaction mixture restores MPP activity, indicating that Triton X-100 dissociates bound subunit IX from the active site of reconstituted MPP. The aromatic group, rather than the hydroxyl group, at Tyr(57) of core I is essential for reconstitutive activity.
Deng
K
K
Department of Biochemistry and Molecular Biology, Oklahoma State University, Stillwater, Oklahoma 74078, USA.
Shenoy
S K
SK
Tso
S C
SC
Yu
L
L
Yu
C A
CA
D000595:Amino Acid Sequence; D000818:Animals; D002417:Cattle; D014450:Electron Transport Complex III / Q000378:metabolism*; D008666:Metalloendopeptidases / Q000737:chemistry / Q000378:metabolism*; D008929:Mitochondria, Heart / Q000201:enzymology*; D008969:Molecular Sequence Data; D017830:Octoxynol / Q000494:pharmacology; D021122:Protein Subunits; D011994:Recombinant Proteins / Q000096:biosynthesis; D013329:Structure-Activity Relationship; D013379:Substrate Specificity; D000096122:Mitochondrial Processing Peptidase
D014450:Electron Transport Complex III; D008666:Metalloendopeptidases; D017830:Octoxynol; D021122:Protein Subunits; D011994:Recombinant Proteins; D000096122:Mitochondrial Processing Peptidase