Investigation of cu(I)-dependent 2,4,5-Trihydroxyphenylalanine Quinone Biogenesis in Hansenula polymorpha Amine Oxidase

Nicole M Samuels,Judith P Klinman

doi:10.1074/jbc.m601501200

Abstract

Copper, a mediator of redox chemistries in biology, is often found in enzymes that bind and reduce dioxygen. Among these, the copper amine oxidases catalyze the oxidative deamination of primary amines utilizing a type(II) copper center and 2,4,5-trihydroxyphenylalanine quinone (TPQ), a covalent cofactor derived from the post-translational modification of an active site tyrosine. Previous studies established the dependence of TPQ biogenesis on Cu(II); however, the dependence of cofactor formation on the biologically relevant Cu(I) ion has remained untested. In this study, we demonstrate that the apoform of the Hansenula polymorpha amine oxidase readily binds Cu(I) under anaerobic conditions and produces the quinone cofactor at a rate of 0.28 h(-1) upon subsequent aeration to yield a mature enzyme with kinetic properties identical to the protein product of the Cu(II)-dependent reaction. Because of the change in magnetic properties associated with the oxidation of copper, electron paramagnetic resonance spectroscopy was employed to investigate the nature of the rate-limiting step of Cu(I)-dependent cofactor biogenesis. Upon aeration of the unprocessed enzyme prebound with Cu(I), an axial Cu(II) electron paramagnetic resonance signal was found to appear at a rate equivalent to that for the cofactor. These data provide strong evidence for a rate-limiting release of superoxide from a Cu(II)(O(2)(.)) complex as a prerequisite for the activation of the precursor tyrosine and its transformation for TPQ. As copper is trafficked to intracellular protein targets in the reduced, Cu(I) state, these studies offer possible clues as to the physiological significance of the acquisition of Cu(I) by nascent H. polymorpha amine oxidase.

Highlights

These properties contrast with Cu(II)-catalyzed cofactor biogenesis, which proceeded at a rate 17-fold faster and with the observation of the putative tyrosinate-Cu(II) ligand-to-metal charge transfer (LMCT) species (350 nm) [6, 7]
Because the decay of the 350-nm intermediate is rate-limiting in Cu(II)-catalyzed trihydroxyphenylalanine quinone (TPQ) biogenesis, the inability to detect this optical intermediate coupled with the diminished value for kTPQ most plausibly indicated a new rate-limiting step further upstream in the Cu(I)-dependent reaction
We have shown that the in vitro reconstitution of apoHPAO with Cu(I) supports TPQ biogenesis, albeit at a rate much slower than the Cu(II)-dependent reaction

Summary

EXPERIMENTAL PROCEDURES

General—Tetrakis(acetonitrile) copper(I) hexafluorophosphate was purchased from Aldrich. The remaining portion of the aerated protein sample was incubated in a 25 °C bath, and aliquots (250 ␮l) were transferred to EPR tubes and frozen at the designated times. Metal Binding Stoichiometry—ApoHPAO in 50 mM HEPES, pH 7.0 (ϳ26 ␮M), was anaerobically reconstituted with an equivalent of stock tetrakis(acetonitrile)copper(I) hexafluorophosphate (700 ␮M in acetonitrile) or CuCl2 (700 ␮M in water) for 1 h and subsequently washed with anaerobic buffer to remove unbound metal. Steady State Kinetic Analysis of HPAO Reconstituted with Copper—ApoHPAO was anaerobically reconstituted with an equivalent of tetrakis(acetonitrile)copper(I) hexafluorophosphate or CuCl2 for 1 h and subsequently allowed to produce TPQ in air-saturated buffer at 25 °C for 20 h. Oxygen consumption was measured under the following conditions: 6 – 8 ␮M HPAO, 5 mM methylamine, 100 mM KPi, 100 mM KCl, pH 7.2, at 25 °C

RESULTS AND DISCUSSION

Equivalent Cua

CONCLUSION