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.
To enable metal affinity purification of cytochrome c oxidase reconstituted into phospholipid vesicles, a histidine-tag was engineered onto the C-terminal end of the Rhodobacter sphaeroides cytochrome c oxidase subunit II. Characterization of the natively processed wildtype oxidase and artificially processed forms (truncated with and without a his-tag) reveals Km values for cytochrome c that are 6-14-fold higher for the truncated and his-tagged forms than for the wildtype. This lowered ability to bind cytochrome c indicates a previously undetected role for the C-terminus in cytochrome c binding and is mimicked by reduced affinity for an FPLC anion exchange column. The elution profiles and kinetics indicate that the removal of 16 amino acids from the C-terminus, predicted from the known processing site of the Paracoccus 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 metal affinity chromatography, significant improvement is observed in proton pumping analysis by the stopped-flow method. The improved kinetic results are attributed to a homogeneous, correctly oriented vesicle population with higher activity and less buffering from extraneous lipids.
Hiser
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Department of Biochemistry and MSU-NIH Mass Spectrometry Facility, Michigan State University, East Lansing, MI 48824-1319, USA.
Mills
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Schall
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Ferguson-Miller
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D000595:Amino Acid Sequence; D000818:Animals; D001665:Binding Sites / Q000235:genetics; D002417:Cattle; D003576:Electron Transport Complex IV / Q000235:genetics / Q000302:isolation & purification / Q000378:metabolism*; D006639:Histidine / Q000235:genetics* / Q000378:metabolism; D007700:Kinetics; D008969:Molecular Sequence Data; D010088:Oxidoreductases / Q000235:genetics / Q000302:isolation & purification / Q000378:metabolism; D010446:Peptide Fragments / Q000235:genetics* / Q000378:metabolism; D010743:Phospholipids / Q000378:metabolism; D011499:Protein Processing, Post-Translational / Q000235:genetics; D017494:Proton Pumps / Q000235:genetics / Q000378:metabolism*; D011994:Recombinant Proteins / Q000138:chemical synthesis / Q000302:isolation & purification / Q000378:metabolism*; D012242:Rhodobacter sphaeroides / Q000201:enzymology / Q000235:genetics; D019032:Spectrometry, Mass, Matrix-Assisted Laser Desorption-Ionization
D010446:Peptide Fragments; D010743:Phospholipids; D017494:Proton Pumps; D011994:Recombinant Proteins; D006639:Histidine; D010088:Oxidoreductases; C117108:cytochrome C oxidase subunit II; D003576:Electron Transport Complex IV