| Abstract
| - Plastocyanin is a small blue copper protein that shuttles electrons as part of the photosyntheticredox chain. Its redox behavior is changed at low pH as a result of protonation of the solvent-exposedcopper-coordinating histidine. Protonation and subsequent redox inactivation could have a role in the downregulation of photosynthesis. As opposed to plastocyanin from other sources, in fern plastocyanin His90protonation at low pH has been reported not to occur. Two possible reasons for that have been proposed: π−π stacking between Phe12 and His90 and lack of a hydrogen bond with the backbone oxygen of Gly36.We have produced this fern plastocyanin recombinantly and examined the properties of wild-type proteinand mutants Phe12Leu, Gly36Pro, and the double mutant with NMR spectroscopy, X-ray crystallography,and cyclic voltammetry. The results demonstrate that, contrary to earlier reports, protonation of His90 inthe wild-type protein does occur in solution with a pKa of 4.4 (±0.1). Neither the single mutants nor thedouble mutant exhibit a change in protonation behavior, indicating that the suggested interactions have noinfluence. The crystal structure at low pH of the Gly36Pro variant does not show His90 protonation, similarto what was found for the wild-type protein. The structure suggests that movement of the imidazole ring ishindered by crystal contacts. This study illustrates a significant difference between results obtained in solutionby NMR and by crystallography.
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