Investigating the cause of the alkaline transition in phytocyanins
The phytocyanins are a family of plant cupredoxins that have been subdivided into the stellacyanins, plantacyanins, and uclacyanins. All of these proteins possess the typical type 1 His2Cys equatorial ligand set at their mononuclear copper sites, but the stellacyanins have an axial Gln ligand in place of the weakly coordinated Met of the plantacyanins, uclacyanins, and most other cupredoxins. The stellacyanins exhibit altered visible, EPR, and paramagnetic 1H NMR spectra at elevated pH values and also modified reduction potentials. This alkaline transition occurs with a pKa of ~10 [Dennison, C., Lawler, A. T. (2001) Biochemistry 40, 3158-3166]. In this study we demonstrate that the alkaline transition has a similar influence on the visible, EPR, and paramagnetic NMR spectra of cucumber basic protein (CBP), which is a plantacyanin. The mutation of the axial Gln95 ligand into a Met in umecyanin (UMC), the stellacyanin from horseradish roots, and the axial Met89 into a Gln in CBP have very limited, yet similar, influence on the pKa for the alkaline transition as judged from alterations in visible spectra. The complete removal of the axial ligand in the Met89Val variant of CBP results in a slightly larger decrease in the pKa for this effect, but similar spectral alterations are still observed at elevated pH. Thus, the axial Gln ligand is not the cause of the alkaline transition in Cu(II) stellacyanins, and alterations in the active site structures of the phytocyanins have a limited effect on this feature. The conserved Lys residue found adjacent to the axial ligand in the sequences of all phytocyanins, and implicated as the trigger for the alkaline transition, has been mutated to an Arg in UMC. The influence of increasing pH on the spectroscopic properties of Lys96Arg UMC is almost identical to those of the wild type protein, and thus, this residue is not responsible for the alkaline transition. However, a positively charged residue in this position seems to be important for the correct folding of UMC. Other possible triggers for the effects seen in the phytocyanins at elevated pH are discussed along with the relevance of the alkaline transition.
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|Item Type:||Journal Article|
|Additional Information:||This article is freely available from the American Chemical Society website 12 months after the publication date. See links to publisher website in this record.|
|Divisions:||Past > QUT Faculties & Divisions > Faculty of Science and Technology|
|Copyright Owner:||Copyright 2005 American Chemical Society|
|Deposited On:||19 Jul 2007 00:00|
|Last Modified:||29 Feb 2012 13:30|
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