Interaction of pyridoxal phosphate modified cytochromes c with mitoplasts

1. The stability of the native conformation of the heme crevice of pyridoxal phosphate (PLP)-ferricytochromes c as assayed by the pK, for 695 nm absorption band varies considerably. The pKa values are 8.76 for cytochrome c modified by PLP at lysine 79[PLP(Lys 79)-cyt. c], 9.23 for cytochrome c modified by PLP at lysine 86 [PLP(Lys 86)-cyt.c], 9.34 for doubly PLP substituted cytochrome c at lysines 79 and 86 [(PLP)2-cyt. c], 9.50 for triply substituted cytochrome c [(PLP)3-cyt. c] and 9.06 for native cytochrome c, which indicates less stable heme crevice of PLP-cytochrome c. 2. The singly PLP-modified cytochrome c indicate decreased activities with mitochondrial cytochrome c oxidase in the following order: PLP(Lys 86)-cyt. c less than PLP(Lys 79)-cyt. c less than native cytochrome c. The high affinity Km for PLP(Lys 86)-cyt. c, PLP(Lys 79)-cyt. c and native cytochrome c are 0.28 microM, 0.16 microM and 0.02 microM respectively. 3. PLP-cytochromes c show decreased binding affinities to fluorescence probes 12-(9-antroyl)-stearic acid and pyrene-labelled mitoplasts. The quenching of singly PLP-modified cytochrome c depends significantly on the ionic strength.