Four-electron reduction of dioxygen by a multicopper oxidase, CueO, and roles of Asp112 and Glu506 located adjacent to the trinuclear copper center

金沢大学理工研究域物質化学系The mechanism of the four-electron reduction of dioxygen by a multicopper oxidase, CueO, was studied based on reactions of single and double mutants with Cys500, a type I copper ligand, and the noncoordinating Asp112 and Glu506, which form hydrogen bonds with the trinuclear copper center directly and indirectly via a water molecule. The reaction of C500S containing a vacant type I copper center produced intermediate I in an EPR-silent peroxide-bound form. The formation of intermediate I from C500S/D112N was restricted due to a reduction in the affinity of the trinuclear copper center for dioxygen. The state of intermediate I was realized to be the resting form of C500S/E506Q and C500S of the truncated mutant Δα5-7CueO, in which the 50 amino acids covering the substrate-binding site were removed. Reactions of the recombinant CueO and E506Q afforded intermediate II, a fully oxidized form different from the resting one, with a very broad EPR signal, g < 2, detectable only at cryogenic temperatures and unsaturated with high power microwaves. The lifetime of intermediate II was prolonged by the mutation at Glu506 involved in the donation of protons. The structure of intermediates I and II and the mechanism of the four-electron reduction of dioxygen driven by Asp112 and Glu506 are discussed. © 2009 by The American Society for Biochemistry and Molecular Biology, Inc

Rotational Energy Barrier around the C1?C11 Single Bond in Lamellarins: A Study by Variable-Temperature NMR

In order to estimate the free energy barrier to rotation around the C1-C11 single bond in lamellarins, new lamellarin analogues (1a), (1b), (2a), and (2b) possessing diastereotopic protons or carbons at the C1 aryl moiety were synthesized. Variable-temperature 1H and 13C NMR measurements of these analogues revealed that the free energy barriers to rotation around the C1-C11 axis in 5,6-saturated and 5,6-unsaturated lamellarins were around 72-74 and 83-87 kJ/mol, respectively

Crucial Fas–Fas ligand interaction in spontaneous acceptance of hepatic allografts in mice

The Fas/Fas ligand (FasL) system plays important roles in the immune system, including host immunoregulation and cytotoxicity. In this study, we investigated the involvement of Fas–FasL interactions in spontaneous acceptance of hepatic allografts in murine orthotopic liver transplantation. Liver transplantation between the C57BL/6 (B6, H-2(b)) donor and the MRL/Mp (MRL, H-2(k)) recipient was performed in various combinations of donor and recipient mice with wild type (+/+), Fas-mutant (lpr) or FasL-mutant (gld) genotypes. The prolongation and spontaneous acceptance of the fully allogeneic grafts in recipients was not observed in either MRL-lpr recipients with B6(+/+) livers or MRL(+/+) recipients with B6-gld livers. Moreover, the serum alanine aminotransferase (ALT) levels and the degree of cell infiltration into hepatic allografts on day 7 after transplantation were inversely correlated with the recipient survival time (in days). The donor-specific cytotoxic T-lymphocyte (CTL) activities of the graft-infiltrating cells (GICs) from MRL-gld recipients with B6(+/+) livers were much lower than those from MRL(+/+) or -lpr recipients on days 5 and 10 after transplantation. However, the CTL activities of the GICs from MRL(+/+) and -gld recipients predominately disappeared by day 15 after transplantation. Furthermore, the anti-donor CTL activities induced in MRL(+/+) recipients were ascribed to CD8(+) cells, and were not mediated by Fas–FasL interactions. These results strongly suggest that the Fas/FasL system plays a critical role for recipient immunoregulation, enabling recipients in accepting hepatic allografts by deletion of the donor-specific T cells, but not for CTL/target cell interaction in MRL(+/+) recipients