a b s t r a c tRedox-induced protonation state changes of the Glu residue in the multicopper oxidases, CueO and bilirubin oxidase (BO), were studied by attenuated total reflectance-Fourier transform infrared spectroscopy. By monitoring IR bands of the carboxylic acid C@O stretch in the wild-type and Gluto-Gln mutant enzymes the Glu506 of CueO (Glu463 of BO) was found to be unprotonated in the oxidised and protonated in the reduced forms. The results provided direct evidence for proton uptake by the Glu, suggesting it plays a key role in the proton donation to the activated oxygen species in the catalytic cycle.

Bilirubin oxidase is a multicopper oxidase containing a type I Cu center to specifically oxidize bilirubin and related substrates and a trinuclear Cu center to perform four-electron reduction of dioxygen as the final electron acceptor. Special attentions have been paid on multicopper oxidases, since this class of enzymes has potential uses as electrocatalyst for biofuel cells and biosensor and catalyst to form a variety of dyes. In addition, bilirubin oxidase has been utilized for the clinical test of liver. In spite of wide attention and potential wide use of the enzyme, structural and functional studies on bilirubin oxidase have been limited.The present study is on the proton transfer mechanism for dioxygen reduction by bilirubin oxidase. We performed mutation at Glu463 located in the hydrogen bond network leading from bulk water to the active site deeply buried inside protein molecule. By singly performing mutation at this amino acid and doubly performing mutation at a Cys ligand to type I Cu center in addition to the mutation at Glu463, we could trap two reaction intermediates, and characterized them in comparisons with those trapped in other multicopper oxidases.We believe this study attracts wide attention from researches studying on biochemistry, bioinorganic chemistry, electrochemistry, biophysics and related fields including bioinspired chemistry, and contributes in the understanding of the four-electron reduction by multicopper oxidases and more widely, transport of protons in protein molecules. According to reasons as above we would like to publish our results in the world wide BBRC. sincerely yours, Takeshi Sakurai Cover LetterProton transport pathway in bilirubin oxidase was mutated Two intermediates in the dioxygen reduction steps were trapped and characterized.A specific glutamate for dioxygen reduction by multicopper oxidases was identified.