The Electron Paramagnetic Resonance Spectrum of Ferricytochrome c and Lysine-Modified Derivatives at Alkaline pH

The heme-associated, pH-induced transition of ferricytochrome c and two lysine-modified derivatives was investigated by absorption and electron paramagnetic resonance (E.P.R.) spectroscopy. The transition from type III to type IV ferricytochrome c (as defined by Theorell, H., and Åkesson, Å.: J. Am. Chem. Soc. 63, 1812 (1941)) produced a new E.P.R. spectrum in frozen solution measured at liquid nitrogen temperature. The measured g values were 3.2 and 2.1 (9.16 GHz). The third component of the expected set of three principal g values for low-spin type IV ferricytochrome c was not observed in this study. In two derivatives with modified lysyl residues, trifluoroacetylated and guanidinated ferricytochrome c, the type III to type IV transition was absent, and instead, at alkaline pH, a brown–red form was produced which had a strong absorption band at about 605 nm. The apparent pK's were 10.3 (trifluoroacetylated) and 9.4 (guanidinated). When an alkaline solution of the guanidinated ferricytochrome was frozen, the E.P.R. spectrum had a set of sharp lines (g values 2.79, 2.21, and 1.76) similar to those observed from low-spin type V ferricytochrome c (pH 14). The alkaline trifluoroacetylated protein gave a similar set of signals but in addition contained the signals observed at neutral pH. These results were interpreted by assuming that, at alkaline pH, a hydroxide ion displaced one of the normally coordinated iron ligands with a temperature-dependent equilibrium between high-spin and low-spin states. Freezing the solution to liquid nitrogen temperature shifted the equilibrium to the low-spin form, and, for the trifluoroacetylated derivative, led to partial recovery of the coordination structure present at about pH 7. The E.P.R. spectrum of the guanidinated cytochrome at neutral pH indicated that the iron electronic structure was essentially identical to type III ferricytochrome c. In contrast, the E.P.R. spectrum of the trifluoroacetylated protein was significantly different, but whether the coordination structure has been geometrically distorted or chemically changed remains to be determined.