Abstract
Maturation processes of dinuclear copper proteins such as tyrosinase, catechol oxidase, and hemocyanin have been a long-standing mystery in copper protein chemistry. Until now, several crystal structures have revealed that these copper proteins share a similar dinuclear copper active site, where each copper ion is ligated by three histidine imidazoles, and binds molecular oxygen in a side-on fashion to form a (µ-η2:η2-peroxido)dicopper(II) species not only as the dioxygen-adduct in oxy-hemocyanins but also as the key reactive intermediate for the hydroxylation of phenols to catechols (phenolase reaction) and the oxidation of catechols to o-quinones (catecholase reaction) in tyrosinases and catechol oxidases. Recently, we have succeeded in determining the high-resolution crystal structures of the recombinant pro-form of yellow koji mold tyrosinase to find the existence of a distinct C-terminal domain containing a –CXXC– unit, that is the common sequence motif of the copper chaperons. Thus, the C-terminal domain apparently acts as a copper chaperon, helping construction of the dinuclear copper active site of tyrosinase. Furthermore, we have found that the proteolytic cleavage of the C-terminal domain from the pro-form (inactive-form) of tyrosinase greatly enhances the tyrosinase activity, thus suggesting that the C-terminal domain also acts as a shielding domain to regulate the enzymatic activity. In fact, overall structure of the pro-form resembles the structure of one of the functional units of octopus hemocyanin (oxygen carrier protein), which also has a similar C-terminal domain prohibiting the monooxygenase activity. On the basis of these results together with the detailed kinetic and spectroscopic analyses, the maturation process of the dinuclear copper proteins is discussed to provide new insights into the regulation mechanism of the dicopper protein functions; dioxygen binding and activation. We have also succeeded in evolving phenolase activity from molluscan and arthropod hemocyanins by treating them with a hydrolytic enzyme or an acid, and demonstrated that the reaction mechanism of their phenolase activity is the same to that of tyrosinase itself, that is the electrophilic aromatic substitution mechanism. Furthermore, we have developed an artificial dicopper protein exhibiting catecholase activity using metallo-β-lactamase, a dinuclear zinc enzyme, as a metal binding platform.
Acknowledgment
The authors gratefully acknowledge the contributions of their co-workers listed in the reference papers, especially, Professor Genji Kurisu of Institute of Protein Research, Osaka University for the kind corporation in X-ray crystal analysis. The authors also thank the Ministry of Education, Culture, Sports, Science and Technology, Japan, the Asahi Glass Foundation, the Mitsubishi Foundation, the Nagase Science and Technology Foundation, and the Shorai Foundation for Science and Technology for their financial supports.
References
N. Fujieda, S. Itoh, in Encyclopedia of Inorganic and Bioinorganic Chemistry, ed. by R. Huber, T. Poulos, K. Wieghardt, John Wiley & Sons, Ltd., Chichester, UK, 2015, pp. 1–8.
Shinobu Itoh 【Award recipient】
Shinobu Itoh received his doctor’s degree in engineering from Osaka University in 1986. He joined the Department of Applied Chemistry of Osaka University as an Assistant Professor (1986–1994) and an Associate Professor (1994–1999). During that period, he spent a year (1987–1988) as a postdoctoral fellow in Professor Teddy G. Traylor’s group at the University of California, San Diego (UCSD). In 1999, he moved to Osaka City University as a full Professor, and came back to Osaka University in 2008. His current research interest is chemical modeling and application of redox active sites in biological systems and development of artificial metalloenzymes.
Nobutaka Fujieda
Nobutaka Fujieda was born in 1978. He received his Ph.D. from Kyoto University in 2006 under the supervision of Prof. Kenji Kano. In 2005–2006, he was a research fellow of the Japan Society for Promotion of Science (JSPS). He then joined Kyoto University Pioneering Research Unit for Next Generation as an Assistant Professor (2006–2009) and moved to Osaka University as an Assistant Professor (2009–present). During that period, he concurrently held a post as Visiting Professor in Prof. Thomas R. Ward’s group at University of Basel (2013–2014). His scientific interest is focused on the exploitation of proteins (and peptides) as metal ligand with application in biotechnology including biocatalysis.
