Direct evidence for the protonation of aspartate-75, proposed to be at a quinol binding site, upon reduction of cytochrome bo(3) from Escherichia coli

Aspartate-75 (D75) was recently suggested to participate in a ubiquinone-bi nding site in subunit I of cytochrome bo(3) from Escherichia coli on the ba sis of a structural model [Abramson, J., Riistama, S., Larsson, G., Jasaiti s, A., Svensson-Ek, M., Laakkonen, L., Puustinen, A., Iwata, S., and Wikstr om, M. (2000) Nat. Struct. Biol, 7 (10), 910-917]. We studied the protonati on state of D75 for the reduced and oxidized forms of the enzyme, using a c ombined site-directed mutagenesis, electrochemical, and FTIR spectroscopic approach. The D75H mutant is catalytically inactive, whereas the more conse rvative D75E substitution has quinol oxidase activity equal to that of the wild-type enzyme. Electrochemically induced FTIR difference spectra of the inactive D75H mutant enzyme show a clear decrease in the spectroscopic regi on characteristic of protonated aspartates and glutamates. Strong variation s in the amide I region of the FTIR difference spectrum, however, reflect a more general perturbation due to this mutation of both the protein and the bound quinone. Electrochemically induced FTIR difference spectra on the hi ghly conservative D75E mutant enzyme show a shift from 1734 to 1750 cm(-1) in direct comparison to wild type. After H/D exchange, the mode at 1750 cm( -1) shifts to 1735 cm(-1). These modes, concomitant with die reduced state of the enzyme, can be assigned to the v(C=O) vibrational mode of protonated D75 and E75, respectively. In the spectroscopic region where signals for d eprotonated acidic groups are expected, band shifts for the v(COO-)(s/as) m odes from 1563 to 1554-1536 cm(-1) and from 1315 to 1335 cm(-1) respectivel y, are found for the oxidized enzyme. These signals indicate that D75 (or E 75 in the mutant) is deprotonated in the oxidized form of cytochrome bo3 an d is protonated upon full reduction of the enzyme. It is suggested that upo n reduction of the bound ubiquinone at the high affinity site, D75 takes up a proton, possibly sharing it with ubiquinol.