C-terminal truncation and histidine-tagging of cytochrome c oxidase subunit II reveals the native processing site, shows involvement of the C-terminus in cytochrome c binding, and improves the assay for proton pumping

Citation
C. Hiser et al., C-terminal truncation and histidine-tagging of cytochrome c oxidase subunit II reveals the native processing site, shows involvement of the C-terminus in cytochrome c binding, and improves the assay for proton pumping, BIOCHEM, 40(6), 2001, pp. 1606-1615
Citations number
38
Language
INGLESE
art.tipo
Article
Categorie Soggetti
Biochemistry & Biophysics
Journal title
BIOCHEMISTRY
ISSN journal
0006-2960 → ACNP
Volume
40
Issue
6
Year of publication
2001
Pages
1606 - 1615
Database
ISI
SICI code
0006-2960(20010213)40:6<1606:CTAHOC>2.0.ZU;2-9
Abstract
TO enable metal affinity purification of cytochrome c oxidase reconstituted into phospholipid vesicles, a histidine-tag was engineered onto the C-term inal end of the Rhodobacter sphaeroides cytochrome c oxidase subunit II. Ch aracterization of the natively processed wildtype oxidase and artificially processed forms (truncated with and without a his-tag) reveals Km values fo r cytochrome c that are 6-14-fold higher for the truncated and his-tagged f orms than for the wildtype. This lowered ability to bind cytochrome c indic ates a previously undetected role for the C-terminus in cytochrome c bindin g and is mimicked by reduced affinity for an FPLC anion exchange column. Th e elution profiles and kinetics indicate that the removal of 16 amino acids from the C-terminus, predicted from the known processing site of the Parac occus denitrificans oxidase, does not produce the same enzyme as the native processing reaction. MALDI-TOF MS data show the true C-terminus of subunit II is at serine 290, three amino acids longer than expected. When the his- tagged form is reconstituted into lipid vesicles and further purified by me tal affinity chromatography, significant improvement is observed in proton pumping analysis by the stopped-flow method. The improved kinetic results a re attributed to a homogeneous, correctly oriented vesicle population with higher activity and less buffering from extraneous lipids.