Effects of axial methionine coordination on the in-plane asymmetry of the heme electronic structure of cytochrome c

Abstract

The paramagnetic susceptibility (χ) tensors of the oxidized forms of thermophile Hydrogenobacter thermophilus cytochrome c ₅₅₂ (Ht cyt c ₅₅₂) and a quintuple mutant (F7A/V13 M/F34Y/E43Y/V78I; qm) of mesophile Pseudomonas aeruginosa cytochrome c ₅₅₁ (Pa cyt c ₅₅₁) have been determined on the basis of the redox-dependent ¹H NMR shift changes of the main-chain NH and C_αH proton resonances of non-coordinated amino acid residues and the NMR structures of the reduced forms of the corresponding proteins (J. Hasegawa, T. Yoshida, T. Yamazaki, Y. Sambongi, Y. Yu, Y. Igarashi, T. Kodama, K. Yamazaki, Y. Kyogoku, Y. Kobayashi (1998) Biochemistry 37:9641–9649; J. Hasegawa, S. Uchiyama, Y. Tanimoto, M. Mizutani, Y. Kobayashi, Y. Sambongi,Y. Igarashi (2000) J Biol Chem 275:37824–37828). From the χ tensors determined, we obtained the contact shifts for heme methyl proton resonances, which provided the heme electronic structures of the oxidized forms of Ht cyt c ₅₅₂ and qm. We also characterized the heme electronic structure of the cyanide adducts of the proteins, where the axial Met was replaced by an exogenous cyanide ion, through the analysis of ¹H NMR spectra. The results indicated that the heme electronic structures of both the proteins in their oxidized forms with axial His and Met coordination are largely different to each other, while those in their cyanide adducts are similar to each other. These results demonstrated that the orientation of the axial Met sulfur lone pair, with respect to heme, predominantly contributes to the spin delocalization into the porphyrin-π system of heme in the oxidized proteins with axial His and Met coordination.