ABSTRACT
The bax-type cytochrome c oxidase from Thermus thermophilus is known as a two subunit enzyme. Deduced from the crystal structure of this enzyme, we discovered the presence of an additional transmembrane helix "subunit IIa" spanning the membrane. The hydrophobic N-terminally blocked protein was isolated in high yield using high-performance liquid chromatography. Its complete amino acid sequence was determined by a combination of automated Edman degradation of both the deformylated and the cyanogen bromide cleaved protein and automated C-terminal sequencing of the native protein. The molecular mass of 3,794 Da as determined by MALDI-MS and by ESI requires the N-terminal methionine to be formylated and is in good agreement with the value calculated from the formylmethionine containing sequence (3,766.5 Da + 28 Da = 3,794.5 Da). This subunit consits of 34 residues forming one helix across the membrane (Lys5-Ala34), which corresponds in space to the first transmembrane helix of subunit II of the cytochrome c oxidases from Paracoccus denitrificans and bovine heart, however, with opposite polarity. It is 35% identical to subunit IV of the ba3-cytochrome oxidase from Natronobacterium pharaonis. The open reading frame encoding this new subunit IIa (cbaD) is located upstream of cbaB in the same operon as the genes for subunit I (cbaA) and subunit II (cbaB).
Subject(s)
Cytochrome b Group/chemistry , Electron Transport Complex IV/chemistry , Thermus thermophilus/chemistry , Amino Acid Sequence , Animals , Cattle , Chromatography, High Pressure Liquid , Cyanogen Bromide/metabolism , Electrophoresis, Polyacrylamide Gel , Methionine/chemistry , Models, Genetic , Models, Molecular , Molecular Sequence Data , Myocardium/enzymology , Open Reading Frames , Protein Structure, Tertiary , Sequence Homology, Amino Acid , Spectrometry, Mass, Electrospray Ionization , Spectrometry, Mass, Matrix-Assisted Laser Desorption-IonizationABSTRACT
Following hints from X-ray data (Ostermeier C et al., 1997, Proc Natl Acad Sci USA 94:10547-10553; Yoshikawa S et al., 1998, Science 280: 1723-1729), chemical evidence is presented from four distantly related cytochrome-c oxidases for the existence of a copperB-coordinated His240-Tyr244) cross-link at the O2-activating Heme Fea3-CuB center in the catalytic subunit 1 of the enzyme. The early evolutionary invention of this unusual structure may have prevented damaging *OH-radical release at e(-)-transfer to dioxygen and thus have enabled O2 respiration.
Subject(s)
Copper/chemistry , Electron Transport Complex IV/chemistry , Histidine/chemistry , Tyrosine/chemistry , Amino Acid Sequence , Animals , Binding Sites , Cattle , Mass Spectrometry , Models, Chemical , Models, Molecular , Molecular Sequence Data , Oxygen Consumption , Paracoccus denitrificans/chemistryABSTRACT
Isoelectric focusing, CD, steady-state and time-resolved fluorescence spectroscopy were used to compare the native recombinant human DNA-repair protein O6-alkylguanine-DNA alkyltransferase (AGT) with AGT derivatives methylated or benzylated on Cys145 or modified by site-directed mutagenesis at the active centre (Met145 mutant). The AGT protein is approximately spherical with highly constrained Trp residues, but is not stabilized by disulphide bridges. In contrast with native AGT, alkylated AGT precipitated at 25 degrees C but remained monomeric at 4 degrees C. As revealed by isoelectric focusing, pI changed from 8.2 (AGT) to 8. 4 (Cys145-methylated AGT) and 8.6 (Cys145-benzylated AGT). The alpha-helical content of the Met145 mutant was decreased by approx. 5% and Trp residues were partially liberated. Although non-covalent binding of O6-benzylguanine did not alter the secondary structure of AGT, its alpha-helical content was increased by approx. 2% on methylation and by approx. 4% on benzylation, altogether indicating a small conformational change in AGT on undergoing alkylation. No signal sequences have been found in AGT that mark it for polyubiquitination. Therefore the signal for AGT degradation remains to be discovered.
