Modified Reactivity toward O-2 in First Shell Variants of Fet3p: Geometric and Electronic Structure Requirements for a Functioning Trinuclear Copper Cluster

作者：Kjaergaard Christian H; Qayyum Munzarin F; Augustine Anthony J; Ziegler Lynn; Kosman Daniel J^*; Hodgson Keith O; Hedman Britt; Solomon Edward I

来源：Biochemistry, 2013, 52(21): 3702-3711.

DOI：10.1021/bi4002826

Multicopper oxidases (MCOs) carry out the most energy efficient reduction of O-2 to H2O known, i.e., with the lowest overpotential. This four-electron process requires an electron mediating type 1 (T1) Cu site and an oxygen reducing trinuclear Cu cluster (TNC), consisting of a binuclear type 3 (T3)- and a mononuclear type 2 (T2) Cu center. The rate-determining step in O-2 reduction is the first two-electron transfer from one of the T3 Cu's (T3 beta) and the T2 Cu, forming a bridged peroxide intermediate (PI). This reaction has been investigated in T3 beta Cu variants of the Fet3p, where a first shell His ligand is mutated to Glu or Gin. This converts the fast two-electron reaction of the wild-type (WT) enzyme to a slow one-electron oxidation of the TNC. Both variants initially react to form a common T3 beta Cu(II) intermediate that converts to the Glu or Gln bound resting state. From spectroscopic evaluation, the nonmutated His ligands coordinate linearly to the T3 beta Cu in the reduced TNCs in the two variants, in contrast to the trigonal arrangement observed in the WT enzyme. This structural perturbation is found to significantly alter the electronic structure of the reduced TNC, which is no longer capable of rapidly transferring two electrons to the two perpendicular half occupied pi*-orbitals of O-2, in contrast to the WT enzyme. This study provides new insight into the geometric and electronic structure requirements of a fully functional TNC for the rate determining two-electron reduction of O-2 in the MCOs.

• 出版日期2013-5-28