Local electrostatic potentials in pyridoxal phosphate labelled horse heart cytochrome c.
The present work shows the application of an optical label pyridoxal phosphate (PLP) for the experimental determination of local electrostatic potentials in singly substituted cytochromes c modified by pyridoxal 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 pKa values of all ionizable groups and the electrostatic potentials in the modified proteins and to analyse their properties. The experimental pKa values for the pyridine nitrogen and phenolic hydroxyl of the bound label were obtained from pH-dependent absorbance and fluorescence measurements, as follows: 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 (fluorescence); 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 fluorescence data are related to differences in the behaviour of the bound label in the ground and excited electronic states and to intermolecular charge-charge interactions. Molecular modelling was used to generate the atomic co-ordinates of the PLP-modified horse heart cytochrome c necessary for the theoretical calculations of the pKa values and electrostatic potentials.
Miteva
M A
MA
Laboratory of Biophysical Chemistry, Bulgarian Academy of Sciences, Sofia, Bulgaria.
Kossekova
G P
GP
Villoutreix
B O
BO
Atanasov
B P
BP
D000818:Animals; D003574:Cytochrome c Group / Q000737:chemistry*; D006736:Horses; D008958:Models, Molecular; D009206:Myocardium / Q000201:enzymology*; D011732:Pyridoxal Phosphate / Q000378:metabolism*; D055672:Static Electricity
D003574:Cytochrome c Group; D011732:Pyridoxal Phosphate