We have the pleasure for submitting our manuscript entitled "Modifications on the
Hydrogen Bond Network by Mutations of Escherichia coli CueO Affect the Process of Proton Transfer to Dioxygen Leading to Peculiar Alterations of Enzymatic Activities" inBiochemical Biophysical Research Communications. CueO is the cuprous oxidase from Escherichia coli and concerns in a system to excrete excess copper to protect E. coli from oxidative damages. Together with laccase and related enzymes, CueO is a multiocopper oxidase and has the type I copper to mediate the electron transfer from substrate and the trinuclear copper center comprised of type II copper and a pair of type III coppers, at which dioxygen is four-electron reduced without forming activated oxygen species. This conversion of dioxygen to water molecule is performed only by terminal oxidases and multicopper oxidases. Therefore, multicopper oxidases are expected to apply to cathodic enzyme for biofuel cells. We have trapped reaction intermediates by preparing mutants and derivatives of multicopper oxidases, which are electron deficient to fully reduce dioxygen and are difficult to relay protons to dioxygen.We revealed that Glu506 in CueO located adjacent to the trinuclear copper center is a key amino acid to relay protons to dioxygen, because this amino is located in the hydrogen bond network leading from the exterior of the CueO molecule to the trinuclear copper center. We performed systematic mutations at Glu506 and Asp507 to study proton relay mechanism in the course of the four-electron reduction of dioxygen by multicopper oxidases. We found that the conserved Glu506 is exchangeable with Asp.The Ala mutation was also functional presumably because one or two water molecules occupied the open space and the alternative proton relay network was constructed in contrast to the Ala mutations in cytochrome oxidases. The hydrogen bond network was also constructed by the Gln mutation, but Gln had not ability to relay protons because the amide group do not cycle between protonated and deprotonated forms.
Cover LetterThe Ile mutation resulted in complete loss of enzymatic activity because an open space to accommodate a water molecule is not formed due to bulkiness in the side chain.Thus the present systematic mutations at Glu506 and also Asp507, which is located in a branch of the hydrogen bond network and was found to play a role to support the proton transfer, unequivocally showed clear results of the proton relay pathway in multicopper oxidase for the first time. Due to special attention to the four-electron reduction of dixoygen by multicopper oxidases, we believe the present study attracts wide attention from researches studying on biochemistry, inorganic chemistry, electrochemistry and related fields, and this is the reason why we would like to publish the present study as rapidly as possible in BBRC.Proton transfer pathway to dioxygen in CueO was identified.Glu506 is the key amino acid to transport proton.The Ala mutation at Glu506 formed a compensatory proton tra...