Further Enhancement of the Thermostability ofHydrogenobacter thermophilusCytochromec₅₅₂

Abstract

Thermophile Hydrogenobacter thermophilus cytochrome c(552) (HT) is a stable protein with denaturation temperatures (T(m)) of 109.8 and 129.7 degrees C for the oxidized and reduced forms, respectively [Uchiyama, S., Ohshima, A., Nakamura, S., Hasegawa, J., Terui, N., Takayama, S. J., Yamamoto, Y., Sambongi, Y., and Kobayashi, Y. (2004) J. Am. Chem. Soc. 126, 14684-14685]. The removal of a single hydroxyl group from the hydrophobic core of HT, through the replacement of a Tyr by Phe, resulted in further elevation of the T(m) value of the oxidized form by approximately 6 degrees C, the T(m) value of the reduced one remaining essentially unaltered. As a result, the redox potential of the mutant with higher stability in the oxidized form exhibited a negative shift of approximately 20 mV relative to that of wild-type HT in an enthalpic manner. These findings indicated that the redox function of a protein can be enthalpically regulated through the stability of the oxidized form by altering the contextual stereochemical packing of hydrophobic residues in the protein interior using protein engineering.

Methionine Ligand Lability in Bacterial Monoheme Cytochromes c: An Electrochemical Study

“…69 Notably, our data are in good general agreement with the magnitude of redox thermodynamic parameters measured by voltammetry utilizing semipermeable membranes for native HT, and mutations affecting electrostatic interactions of the N- and C- termini. 55,72,73 The comparison indicates that the M61 mutations have not overwhelmingly affected the intrinsic thermodynamics associated with the electron transfer for Ht cyt c .…”

Change in structure and ligand binding properties of hyperstable cytochrome c555 from Aquifex aeolicus by domain swapping

“…Proteins in the cytochrome c (cyt c ) protein family have been studied extensively in terms of protein folding and stability . Met80 and His18 are coordinated to the heme iron in cyt c , creating a relatively high redox potential for electron transfer.…”

“…18,19 Proteins in the cytochrome c (cyt c) protein family have been studied extensively in terms of protein folding and stability. [20][21][22][23][24][25][26][27][28][29][30][31][32][33][34][35] Met80 and His18 are coordinated to the heme iron in cyt c, creating a relatively high redox potential for electron transfer. The redox potential of M80A cyt c was lower than that of wildtype cyt c, due to the loss of the Met80-heme iron coordination bond, 36 whereas M80A cyt c exhibited unusual O 2 and CO binding properties.…”

Correlation between the Stability and Redox Potential of Three Homologous Cytochromescfrom Two Thermophiles and One Mesophile

“…1; their root mean square deviation values are within 1 Å ), but the stability of their oxidized forms against thermal and GdnHCl-induced denaturation are significantly different. 6) The thermal stability of reduced HT c 552 and PA c 551 has already been measured, the denaturation temperatures being over 100 C, 7,8) and their E m values have been determined. PH c 552 , which exhibits intermediate stability among the three proteins in the oxidized state, has not been characterized yet in terms of the stability of the reduced form or of electrochemistry.…”