An engineered CuA Amicyanin capable of intermolecular electron transfer reactions.
The type I copper center of amicyanin was replaced with a binuclear CuA center. To create this model CuA protein, a portion of the amino acid sequence that contains three of the ligands to the native type I copper center of Paracoccus denitrificans amicyanin was replaced with the corresponding portion of sequence that provides five ligands for the CuA center of cytochrome c oxidase from P. denitrificans. UV-visible and electron paramagnetic resonance spectroscopy confirm that the engineered protein as isolated possesses the mixed-valence Cu1.5Cu1.5 (purple) CuA center. Comparison of the spectroscopic properties of this CuA amicyanin with those of the CuA centers of other natural and engineered CuA proteins suggests that the spectroscopic features may be dictated more by the protein host than the sequence of the CuA loop. Novel reactions for a simple CuA model protein are also described. In contrast to other natural and engineered CuA proteins, the fully reduced CuA amicyanin may be reoxidized by molecular oxygen to the mixed-valence state. It is also shown that CuA amicyanin can serve as an electron donor and an electron acceptor for other redox proteins. The mixed-valence form accepts electrons from cytochromes c-551i and c-550 from P. denitrificans. The fully reduced form donates electrons to native and P94F amicyanin. The function as either an electron donor or acceptor is consistent with the measured redox potential of CuA amicyanin of +273 mV. These data indicate that this CuA amicyanin will be a particularly useful model protein for structure-function studies of reactivity and the electron transfer properties of the CuA redox center.
Jones
Limei H
LH
Department of Biochemistry, University of Mississippi Medical Center, 2500 N. State Street, Jackson, MS 39216-4505, USA.
Liu
Aimin
A
Davidson
Victor L
VL
D000595:Amino Acid Sequence; D001426:Bacterial Proteins / Q000737:chemistry* / Q000378:metabolism; D003300:Copper / Q000737:chemistry*; D004579:Electron Transport; D008667:Metalloproteins / Q000737:chemistry; D010084:Oxidation-Reduction; D010231:Paracoccus denitrificans / Q000737:chemistry; D015202:Protein Engineering; D013057:Spectrum Analysis
D001426:Bacterial Proteins; D008667:Metalloproteins; C017079:mauC protein, Methylobacterium extorquens; D003300:Copper