Using Matrix-Assisted Laser Desorption Ionization Mass Spectrometry To Map the Quinol Binding Site of Cytochrome bo₃ from Escherichia coli

Abstract

The cytochrome bo3 ubiquinol oxidase contains at least one and possibly two binding sites for ubiquinol/ubiquinone. Previous studies used the photoreactive affinity label 3-[3H]azido-2-methyl-5-methoxy-6-geranyl-1,4-benzoquinone (azido-Q), a substrate analogue, to demonstrate that subunit II contributes to at least one of the quinol binding sites. In the current work, mass spectroscopy is used to identify a peptide within subunit II that is photolabeled by the azido-Q. Purified cytochrome bo3 was photolabeled as previously described using azido-Q that was not tritiated (i.e., not radiolabeled). Subunit II was then isolated from an SDS-PAGE gel and proteolyzed in situ with trypsin. The resulting peptides were eluted from the gel and then identified using matrix-assisted laser desorption ionization mass spectrometry. The resulting mass spectrum was compared to that obtained by analysis of subunit II that had not been exposed to the photolabel. Using the amino acid sequence, each peak in the mass spectrum of the unlabeled subunit II could be assigned to an expected trypsin fragment. Two additional peaks were observed in the mass spectrum of the photolabeled subunit with m/z 1931.9 and 2287.7. Subtraction of the mass of azido-Q from the peak at m/z 1931.9 results in a mass equivalent to that of a peptide consisting of amino acids 165-178. The assignment of the peak at m/z 2287.7 cannot be made unequivocally and may correspond either to the covalent attachment of azido-Q to peptide 254-270 or to a peptide resulting from incomplete proteolysis. The labeled peptide, 165-178, is within the water-soluble domain of subunit II, whose X-ray structure is known. This peptide is located near the site where CuA is located in the homologous cytochrome c oxidases and can be placed near the interface between subunits I and II.

Elucidating mechanisms in haemcopperoxidases: The high-affinity QHbinding site in quinol oxidase as studied by DONUT-HYSCOREspectroscopy and density functional theory

“…16 Wild-type bo 3 enzyme with bound UQ 8 was purified as described previously. 29 Native UQ 8 was removed by purification of QOX using N,N-dimethyldodecylamine N-oxide (LDAO) as the detergent, followed by detergent exchange with n-dodecyl-b-D-maltoside (b-DM). Reconstitution with exogenous 2,3-dimethoxy-5,6-dimethyl-1,4-benzoquinone (DDQ, see Fig.…”

“…22 Variant cytochrome bo 3 ubiquinol oxidase from E. coli containing one equivalent of bound UQ 8 was purified in n-dodecyl-b-D-maltoside according to the method described previously, 28 and concentrated to approximately 0.1 mM in 100 mM K-Phosphate buffer at pH 8. The presence or absence of ubiquinone was confirmed by FTIR spectroscopy as reported in 31 (data not shown).…”

“…[1][2][3] It catalyses both the two-electron oxidation of ubiquinol-8 (UQ 8 ) and the four-electron reduction of dioxygen. The presence of up to demonstrated that this semiquinone radical in QOX forms a hydrogen bond with a nitrogen atom either from the polypeptide backbone of the protein 16 or from an arginine side chain 17 and very recently studies of Lin at al.…”

“…19 (B), D75 is shown together with the residues suggested to participate in quinone/quinol binding and proton translocation. (C) Chemical structures of (i) the native ubiquinone-8 (UQ 8 ) and of (ii) 2,3-dimethoxy-5,6-dimethyl-1,4-benzoquinone (DDQ).…”

C-terminal Periplasmic Domain of Escherichia coli Quinoprotein Glucose Dehydrogenase Transfers Electrons to Ubiquinone

“…Furthermore, using depth-dependent fluorescent ubiquinone analogues, Miyoshi et al (17) demonstrated that the ubiquinone-reduction site of GDH is located close to the membrane surface rather than in the hydrophobic interior. X-ray crystallographic structures of cytochrome bo in E. coli (18,19) and cytochrome bc 1 complex (Q o and Q i centers) in bovine heart mitochondria have recently been determined (20, 21), and it has been indicated that their ubiquinone-binding sites may be close to the membrane surface. Thus, it seems reasonable that the ubiquinone-reacting site of GDH is located near the membrane surface.…”