Sotaro Fujii
Hiroshima University, Japan
Title: Stability and function of a thermophilic cytochrome c’
Biography
Biography: Sotaro Fujii
Abstract
Cytochromes c' are classifi ed as heme proteins found in restricted Gram-negative bacteria. Th ey usually form a homo dimeric
structure, and the single subunit typically consists of four helix bundle. Biochemical analysis showed that they can bind diatomic
gasses such as NO or CO, but not O2. Recently we purifi ed cytochrome c' from thermophilic Hydrogenophilus thermoluteolus, and
named it PHCP. H. thermoluteolus grows optimally at 52°C, indicating that PHCP is more stable than homologous proteins from
mesophiles. In this study, we compared stability and function of PHCP with its mesophilic homologue, Allochromatium vinosum
cytochrome c' (AVCP) having 55 % amino acid sequence identity. In order to check the stability, we measured the circular dichroism
spectra with increasing temperature. Th e denaturation temperature of PHCP was 87°C, which was higher than that of AVCP
(52°C). Th e X-ray structure comparison between PHCP and AVCP revealed that the stability diff erence was due to the heme-related
interactions and subunit-subunit interactions, which was also proofed by mutagenesis study. Th ese results indicated that PHCP
advantageously retains the native structure at high temperature. Th e PHCP X-ray structure further revealed a ligand binding channel
and a penta-coordinated heme, as observed in the AVCP protein, indicating PHCP could bind diatomic gasses at high temperature.
Th us, we measured the gas binding affi nity of PHCP and AVCP using absorption spectra. Th e association constant (Ka) of PHCP
with CO was 3 times lower than that of AVCP at 25°C, and PHCP could maintain normal spectral changes up to 60°C. In AVCP, such
spectral changes with CO could not to be detected at 60°C, because of denaturation of AVCP. In conclusion, PHCP has a structure
fulfi lling the requirement for both gas-binding function and thermal stability. Th is stable cytochrome c' will become a model for
protein engineering fi eld.