An Engineered CuA Amicyanin Capable of Intermolecular Electron Transfer Reactions

Limei H Jones,Aimin Liu,Victor L Davidson

doi:10.1074/jbc.m308863200

Limei H Jones, Aimin Liu + Show 1 more

Open Access

https://doi.org/10.1074/jbc.m308863200

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Abstract

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.

Highlights

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
CuA amicyanin is unique for an engineered CuA protein in that it may be isolated in the active mixed-valence purple form
The soluble CuA binding domain of P. denitrificans c oxidase (COX) that is expressed in E. coli is isolated in a form that must be denatured, refolded, and reconstituted with copper to yield the holoprotein [12]. Another CuA amicyanin formed by introduction of the sequence from the CuA binding mutant of cytochrome o-quinol oxidase into Thiobacillus versutus amicyanin was isolated as a holoprotein [49]

Summary

Reactivity of a New CuA Amicyanin

CuA amicyanin was purified from the periplasmic fraction, which was prepared from E. coli cells as described previously by a lysozyme-osmotic shock procedure [40]. The Em value of CuA amicyanin was determined by spectrochemical titration. The reaction mixture contained 90 ␮M CuA amicyanin in 0.01 M Tris-HCl, pH 7.0, 25 °C. Reduced CuA amicyanin may be reoxidized by molecular oxygen to the mixed-valence purple state. Such a reaction has not been described previously for a natural or engineered CuA protein. It is shown that CuA amicyanin can serve as an electron donor and an electron acceptor for other redox proteins including the natural redox partner for native amicyanin, cytochrome c-551i from P. denitrificans, and the natural redox partner of COX, cytochrome c-550 from P. denitrificans. These data indicate that this CuA amicyanin will be a useful model protein for structure-function studies of reactivity and the electron transfer properties of the CuA redox center

EXPERIMENTAL PROCEDURES

RESULTS

Absorption bands

DISCUSSION