LOCAL ELECTROSTATIC POTENTIALS IN PYRIDOXAL-PHOSPHATE LABELED HORSE HEART CYTOCHROME-C

The present work shows the application of an optical label pyridoxal p hosphate (PLP) for the experimental determination of local electrostat ic potentials in singly substituted cytochromes c modified by pyridoxa l phosphate at Lys 79 (PLP-Lys-79-cyt.c) or at Lys 86 (PLP-Lys-86-cyt. c). PLP has also been used to calculate the pK(a) values of all ioniza ble groups and the electrostatic potentials in the modified proteins a nd to analyse their properties. The experimental pK(a) values for the pyridine nitrogen and phenolic hydroxyl of the bound label were obtain ed from pH-dependent absorbance and fluorescence measurements, as foll ows: in PLP-Lys-79-cyt.c for pyridine nitrogen 4.5 (absorbance) and 5. 1 (fluorescence), for phenolic hydroxyl 8.6 (absorbance) and 8.3 (fluo rescence); in PLP-Lys-86-cyt.c for pyridine nitrogen 4.7 (absorbance) and 5.8 (fluorescence), for phenolic hydroxyl 8.3 (absorbance) and 8.5 (fluorescence). The differences between absorbance and Auorescence da ta are related to differences in the behaviour of the bound label in t he ground and excited electronic states and to intermolecular charge-c harge interactions. Molecular modelling was used to generate the atomi c co-ordinates of the PLP-modified horse heart cytochrome c necessary for the theoretical calculations of the pK(a) values and electrostatic potentials.