USING MATRIX-ASSISTED LASER-DESORPTION IONIZATION MASS-SPECTROMETRY TO MAP THE QUINOL BINDING-SITE OF CYTOCHROME BO(3) FROM ESCHERICHIA-COLI

The cytochrome bo? ubiquinol oxidase contains at least one and possibl y two binding sites for ubiquinol/ubiquinone. Previous studies used th e photoreactive affinity label zido-2-methyl-5-methoxy-6-geranyl-1,4-b enzoquinone (azido-Q), a substrate analogue, to demonstrate that subun it II contributes to at least one of the quinol binding sites. In the current work, mass spectroscopy is used to identify a peptide within s ubunit II that is photolabeled by the azido-Q. Purified cytochrome bo( 3) 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 p eptides were eluted from the gel and then identified using matrix-assi sted 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 ob served in the mass spectrum of the photolabeled subunit with mit 1931. 9 and 2287.7, Subtraction of the mass of azido-Q from the peak at mit 1931.9 results in a mass equivalent to that of a peptide consisting of amino acids 165-178. The assignment of the peak at mit 2287.7 cannot be made unequivocally and may correspond either to the covalent attach ment of azido-Q to peptide 254-270 or to a peptide resulting from inco mplete proteolysis. The labeled peptide, 165-178, is within the water- soluble domain of subunit II, whose X-ray structure is known. This pep tide is located near the site where Cu-A is located in the homologous cytochrome c oxidases and can be placed near the interface between sub units I and II.