The effect of surface charge on the reduction potential and heme propionate ionization of a monoheme cytochrome: spectroscopic and potentiometric studies of trifluoroacetylated cytochrome c(551) from Pseudomonas aeruginosa

摘要

The pH dependence of the mid-point reduction potential (E-m) of Pseudomonas aeruginosa cytochrome c(551) in which the lysine residues had been converted into N-e-trifluoroacetyl-lysines was determined by the method of mixtures and found to be affected by ionizations in both the oxidized and reduced forms with pK(a), values of 6.6 +/- 0.1 and 8.6 +/- 0.1 respectively. Characterization of the ionizations by NMR spectroscopy and UV-Vis spectrophotometry showed that the group ionizing is a heme propionate substituent, similar to the native cytochrome c(551) in which a heme propionate has a redox-dependent ionization with pK(a), values for the oxidized and reduced forms of 6.2 +/- 0.1 and 7.3 +/- 0.1 respectively. At low pH, when the heme propionate is protonated in both redox states of the protein, modification of the surface amine charges causes a 45 +/- 3 mV drop in the value of E-m, an average of 5 mV per surface charge. By contrast, ionization of the heme propionate causes a decrease in E-m, of 65 +/- 3 mV. This demonstrates that a single partly buried charge has a greater effect than nine charges located on the protein surface. (C) 1998 Elsevier Science S.A. All rights reserved. [References: 40]