Characterization of Arabidopsis thaliana Stellacyanin: A Comparison with Umecyanin

Abstract

The cupredoxin domain of a putative type 1 blue copper protein (BCB) from Arabidopsis thaliana was overexpressed and purified. A recursive polymerase chain reaction method was used to synthesize an artificial coding region for the cupredoxin domain of horseradish stellacyanin (commonly known as umecyanin), prior to overexpression and purification. The recombinant proteins were refolded from inclusion bodies and reconstituted with copper, and their in vitro characteristics were studied. Recombinant umecyanin, which is nonglycosylated, has identical spectroscopic and redox properties to the native protein. The UV/Vis and EPR spectra of recombinant BCB and umecyanin demonstrate that they have comparable axial type 1 copper binding sites. Paramagnetic 1H NMR spectroscopy highlights the similarity between the active site architectures of BCB and umecyanin. The reduction potential of recombinant BCB is 252 mV, compared to 293 mV for recombinant umecyanin. Identical pKa values of 9.7 are obtained for the alkaline transitions in both proteins. This study demonstrates that BCB is the A. thaliana stellacyanin and the results form the biochemical basis for a discussion of BCB function in the model vascular plant. © 2004 Wiley-Liss, Inc.