Protonation/deprotonation reactions triggered by photoactivation of photoactive yellow protein from Ectothiorhodospira halophila.
Light-dependent pH changes were measured in unbuffered solutions of wild type photoactive yellow protein (PYP) and its H108F and E46Q variants, using two independent techniques: transient absorption changes of added pH indicator dyes and direct readings with a combination pH electrode. Depending on the absolute pH of the sample, a reversible protonation as well as a deprotonation can be observed upon formation of the transient, blue-shifted photocycle intermediate (pB) of this photoreceptor protein. The latter is observed at very alkaline pH, the former at acidic pH values. At neutral pH, however, the formation of the pB state is not paralleled by significant protonation/deprotonation of PYP, as expected for concomitant protonation of the chromophore and deprotonation of Glu-46 during pB formation. We interpret these results as further evidence that a proton is transferred from Glu-46 to the coumaric acid chromophore of PYP, during pB formation. One cannot exclude the possibility, however, that this transfer proceeds through the bulk aqueous phase. Simultaneously, an amino acid side chain(s) (e.g. His-108) changes from a buried to an exposed position. These results, therefore, further support the idea that PYP significantly unfolds in the pB state and resolve the controversy regarding proton transfer during the PYP photocycle.
Hendriks
J
J
Laboratory for Microbiology, E. C. Slater Institute, BioCentrum, University of Amsterdam, 1018 WS Amsterdam, The Netherlands.
Hoff
W D
WD
Crielaard
W
W
Hellingwerf
K J
KJ
D001426:Bacterial Proteins / Q000737:chemistry* / Q000235:genetics; D006863:Hydrogen-Ion Concentration; D008969:Molecular Sequence Data; D016297:Mutagenesis, Site-Directed; D009154:Mutation; D010777:Photochemistry; D020283:Photoreceptors, Microbial / Q000737:chemistry*; D011487:Protein Conformation; D017510:Protein Folding; D011522:Protons; D012244:Rhodospirillaceae / Q000378:metabolism*; D013053:Spectrophotometry
D001426:Bacterial Proteins; D020283:Photoreceptors, Microbial; D011522:Protons; C050162:photoactive yellow protein, Bacteria