H-1 AND B-11 NMR EVIDENCE FOR SPECIFIC BINDING OF BORATE ION TO CYTOCHROME-C

Cytochrome c possesses a positively charged surface which binds specif ically to mitochondria enzymes and metabolites. The affinity of the cy tochrome c surface to charged ligands is demonstrated by specific bind ing to berate ions. The effect of the ionic bond formed between the be rate and the cytochrome is observed in both H-1 and B-11 NMR spectra. New narrow peaks with prolonged T-1 values appear in the B-11 spectrum and are assigned to the bound tetrahedral berate ion. In the H-1 spec trum changes are observed in the peaks of the iron ligand histidine 18 beta and the heme methyls 3 and 8. These changes are affected by the ionic strength of the solution. The exchange process between the free and berate-bound forms of the protein is established by a two-dimensio nal exchange spectroscopy experiment. The k(diss) for the process is 2 4 +/- 2 s(-1), as measured at 5 degrees C by an inversion transfer exp eriment. K-D values were calculated at several temperatures and range from 67 +/- 7 mM at 2.7 degrees C to 143 +/- 14 mM at 14 degrees C. (C ) 1998 Elsevier Science S.A. All rights reserved.