Mass Spectrometric Analysis of the Ubiquinol-binding Site in Cytochrome bd from Escherichia coli

Daisuke Fujita,Yushi Matsumoto,Masatoshi Murai,Tatsushi Mogi,Hideto Miyoshi,Masasuke Yoshida,Kimitoshi Sakamoto

doi:10.1074/jbc.m508206200

Abstract

Cytochrome bd is a heterodimeric terminal ubiquinol oxidase in the aerobic respiratory chain of Escherichia coli. For understanding the unique catalytic mechanism of the quinol oxidation, mass spectrometry was used to identify amino acid residue(s) that can be labeled with a reduced form of 2-azido-3-methoxy-5-methyl-6-geranyl-1,4-benzoquinone or 2-methoxy-3-azido-5-methyl-6-geranyl-1,4-benzoquinone. Matrix-assisted laser desorption ionization time-of-flight mass spectrometry demonstrated that the photo inactivation of ubiquinol-1 oxidase activity was accompanied by the labeling of subunit I with both azidoquinols. The cross-linked domain was identified by reverse-phase high performance liquid chromatography of subunit I peptides produced by in-gel double digestion with lysyl endopeptidase and endoproteinase Asp-N. Electrospray ionization quadrupole time-of-flight mass spectrometry determined the amino acid sequence of the peptide (m/z 1047.5) to be Glu(278)-Lys(283), where a photoproduct of azido-Q(2) was linked to the carboxylic side chain of I-Glu(280). This study demonstrated directly that the N-terminal region of periplasmic loop VI/VII (Q-loop) is a part of the quinol oxidation site and indicates that the 2- and 3-methoxy groups of the quinone ring are in the close vicinity of I-Glu(280).

Highlights

Nol-8 (Q8H2),2 leading to the release of four protons from quinols to the periplasm
Monophasic decay of the oxidase activity indicates the labeling of the quinol oxidation site with a single azido-Q2H2 molecule
These results indicate that the photoaffinity labeling of azido-Q2H2 to the quinol-binding site inactivates the quinol oxidase activity of cytochrome bd

Summary

EXPERIMENTAL PROCEDURES

Purification of Cytochrome bd and Quinol Oxidase Assay—The enzyme was isolated from the cytochrome bd-overproducing strain GR84N/pNG2 [34], as described previously [14]. Gel pieces containing subunit I band were washed three times with 1 ml of 70% (v/v) acetonitrile, dried in a vacuum centrifuge, and rehydrated with 0.l ml of the digestion buffer. The gel pieces were first incubated with Lep, washed three times with 1 ml of 70% (v/v) acetonitrile, and treated with Asp-N or CNBr as described above. MALDI-TOF MS—The samples were analyzed on an AXIMA-CFR plus (Shimadzu Co., Kyoto) TOF mass spectrometer in a linear (for proteins) or reflection (for peptides) mode with positive ion detection. Reverse-phase HPLC—Subunit I peptides (100 ␮g) produced by double digestion with Lep and Asp-N were separated by reverse-phase HPLC on a Develosil 300C8-HG-5 column (4.6-mm inner diameter ϫ 15 cm; Nomura-Kagaku Co.) using a gradient formed from 0.1% acetic acid and 30% acetonitrile containing 0.1% acetic acid with a flow rate of 1 ml/min. The nomenclature used for fragment ions was N-terminal CϭO-containing fragments (b type) and C-terminal N-H-containing fragments (y type), which were derived by the cleavage at the middle of peptide bonds [39]

RESULTS

DISCUSSION

Cytochrome bd is widely distributed as the terminal oxidase from