High Thermal Stability and Unique Trimer Formation of Cytochromec′ from ThermophilicHydrogenophilus thermoluteolus

Abstract

Sequence analysis indicated that thermophilic Hydrogenophilus thermoluteolus cytochrome c' (PHCP) and its mesophilic homolog, Allochromatium vinosum cytochrome c' (AVCP), closely resemble each other in a phylogenetic tree of the cytochrome c' family, with 55% sequence identity. The denaturation temperature of PHCP was 87 °C, 35 °C higher than that of AVCP. Furthermore, PHCP exhibited a larger enthalpy change value during its thermal denaturation than AVCP. While AVCP was dimeric, as observed previously, PHCP was trimeric, and this was the first observation as a cytochrome c'. Dissociation of trimeric PHCP and its protein denaturation reversibly occurred at the same time in a two-state transition manner. Therefore, PHCP is enthalpically more stable than AVCP, perhaps due to its unique trimeric form, in addition to the lower number of Gly residues in its putative α-helical regions.

High stability of apo-cytochrome c’ from thermophilic Hydrogenophilus thermoluteolus

“…This value was lower than that of holo-PHCP (87.1°C), but close to that obtained for the holo-AVCP protein under the same conditions (52.4°C). 3) These results indicate that heme contributes to the PHCP stability, as observed for AA cytc 555 , 12) but that apo-PHCP is stable enough against thermal denaturation, the level being similar to that of mesophilic holo-AVCP.…”

“…In addition, PHCP appears to be homotrimeric unlike other usual dimeric cytochromes c', and is more stable than its mesophilic counterpart, Allochromatium vinosum cytochrome c' (AVCP), exhibiting 55% sequence identity. 3) These unique features of PHCP were found as the holoprotein that covalently binds heme.…”

“…3) The apo-forms of PHCP and AVCP were prepared by the modified method of Fisher et al 5) which had been applied to class I cytochromes c, as described previously. 7) Ten mg of Ag 2 SO 4 in 0.1 mL of ultrapure water and 80 μL of acetic acid were added to 0.8 mL aliquots of 0.3-0.5 mM purified holo-PHCP (in the presence of 8 M urea) and holo-AVCP (without urea) solutions.…”

“…The raw CD data for the thermal denaturation of apo-PHCP could be normalized and subjected to nonlinear least-squares fitting ( Fig. 2(C)) as described previously for the holoprotein, 3) which showed midpoint of thermal denaturation of 50.3 ± 1.7°C. This value was lower than that of holo-PHCP (87.1°C), but close to that obtained for the holo-AVCP protein under the same conditions (52.4°C).…”

Thermal stability of cytochrome c′ from mesophilic Shewanella amazonensis

“…PHCP was thermally more stable than AVCP, the denaturation temperatures under the same experimental conditions being 87 and 52°C, respectively. 3) We searched for another suitable cytochrome c′ for a further protein thermal stability study involving homologous cytochromes c′. We found a gene encoding a cytochrome c′ protein (which we here denote as SACP) from mesophilic Shewanella amazonensis, growing optimally at 37°C, 4) which is higher and lower, respectively, than the optimal growth temperatures of A. vinosum and H. thermoluteolus.…”