Roleof Hydrogen Bond Connecting Ligands for Substrate and Type I Copper in Copper(I) Oxidase CueO Kunishige Kataoka and Takeshi Sakurai* Graduate School of Natural Science and Technology, Kanazawa University, Kakuma, Kanazawa, Ishikawa 920-1192 (Received May 3, 2013; CL-130422; E-mail: tsakurai@se.kanazawa-u.ac.jp) Mutational and kinetic studies on Asp439, a ligand for the substrate-binding site in the copper(I) oxidase®CueO®and the deletion mutant®¦¡57 CueO®indicated that the electron transfer from the copper(I) ion as the substrate to the type I copper is performed though the hydrogen bond between Asp439 and His443, a ligand for the type I copper, and also by means of the through-space mechanism directly. CueO is a multicopper oxidase (MCO) containing a type I (T1) copper, a type II (T2) copper, and a pairof type III (T3) coppers in the active site, and it functions as copper(I) oxidase in the copper efflux system of Escherichia coli (E. coli). 1,2 T1 copper functions as the site to oxidize copper(I) ion to the less toxic copper(II) ion by mediating the electron transfer between the substrate and the trinuclear copper center (TNC) comprising T2 and T3 coppers, where dioxygen is reduced to water without forming an activated oxygen species. 39 The substrate-binding site (copper-binding site) and T1 copper are connected with a hydrogen bond between Asp439 and His443 (Figure 1). 8 Analogous hydrogen bonds to connect the substrate-binding site and T1 copper have also been found in ferroxidases such as ceruloplasmin 10 and Fet3p. 11 These hydrogen bonds have been considered to function as a pathway of electron transfer, 8,10,11 although the through-space mechanism treads the shortest distance, i.e., 0.75 nm in the case of CueO. In a previous study, we performed the Asp439 to Ala mutation of CueO and the deletion mutant, i.e., ¦¡57 CueO, in which 50 amino acids covering the substrate-binding site were removed from the parent CueO. 6,8 The redox potential of T1 copper was shifted 5070 mV toward the positive direction as a resultof decreasing the donating ability of His443 to T1 copper without inducing destabilization of the T1 copper center. 6 Concomitantly, the oxidizing activities of organic substrates were newly expressed or enhanced, showing increases in the oxidizing activity of 2,2¤-azinobis(3-ethylbenzothiazoline-6-sul- fonic acid) (ABTS) by 41-fold and 4-fold from those of CueO and ¦¡57 CueO, respectively. In the present study, we determined the oxidizing activities of the Asp439Ala mutants of CueO and ¦¡57 CueO toward copper(I) ion, ABTS, and 1,3-dimethoxy-2-hydroxybenzene (DMP). Oxidizing activities of ABTS were determined both in the presence and absence of copper(II) ions, and those of DMP were determined in the presence of copper(II) ions because it has been discovered that the Cu(II) ion bound to the substrate-binding site mediates the electron transfer between organic substrates and T1 copper. 6,12 Further, we determined affinities of the copper-binding site for Cu(I) and Cu(II) ions and affinities of TNC for O 2 to evaluate the effect of deleting the hydrogen bond on the enzymatic activities of CueO and derivatives. The present results show that the hydrogen bond functions as the pathway of electron transfer between the substrate-binding site and T1 copper in CueO. The genes for the Asp439Ala mutants of CueO and ¦¡57 CueO were prepared using a QuikChange kit (Stratagene). E. coli BL21(DE3) was transformed with the mutant plasmids and cultivated. Purifications of mutants were conducted as described previously. 59 The Asp439 mutants of CueO and ¦¡57 CueO contained four copper ions within experimental errors of 5%, as determined by atomic absorption spectroscopy, and gave absorption, circular dichroism (CD), and electron spin resonance (EPR) spectra similar to the parent enzymes (See Supporting Information, S1). 6,8,13 Copper(I)-oxidizing activities of the parent CueO and ¦¡57 CueO and the Asp439Ala mutants have been determined from consumptions of dioxygen (Table 1, Figure 2, and Sup- porting Information, S2). 13 The k cat values of the Asp439Ala mutants were decreased to ca. 50%from those of CueO and ¦¡57 CueO, while the redox potential of T1 copper was shifted to positive direction by 70 and 50mV, respectively 8 (The K m and k cat values of CueO in ref 14 exhibited the lower affinity and activity for Cu(I) ions compared to the present results). In spite of the change in the driving force, the electron transfer from Cu(I) ion to T1 copper has become unfavorable. This fact Figure 1. Superimposition of the structures of CueO (A) (yellow, PDB entry 1N68) and ¦¡57 CueO (blue, PDB entry 2YXV) and the active site of CueO (B) (PDB entry 1N68). Red sphere shows the Cu(II) ion bound at the substrate-binding site, blue sphere T1 copper, green sphere T2 copper, and orange spheres T3 coppers. The very flexible segment to connect between the short ¡-helices 6 and 7 is not seen. Published on the web June 8, 2013 1102 doi:10.1246/cl.130422 © 2013 The Chemical Society of Japan Chem. Lett. 2013, 42, 11021104 www.csj.jp/journals/chem-lett/