Structural and functional insights into thermally stable cytochrome c′ from a thermophile

Abstract

Thermophilic Hydrogenophilus thermoluteolus cytochrome c′ (PHCP) exhibits higher thermal stability than a mesophilic counterpart, Allochromatium vinosum cytochrome c′ (AVCP), which has a homo‐dimeric structure and ligand‐binding ability. To understand the thermal stability mechanism and ligand‐binding ability of the thermally stable PHCP protein, the crystal structure of PHCP was first determined. It formed a homo‐dimeric structure, the main chain root mean square deviation (rmsd) value between PHCP and AVCP being 0.65 Å. In the PHCP structure, six specific residues appeared to strengthen the heme‐related and subunit–subunit interactions, which were not conserved in the AVCP structure. PHCP variants having altered subunit–subunit interactions were more severely destabilized than ones having altered heme‐related interactions. The PHCP structure further revealed a ligand‐binding channel and a penta‐coordinated heme, as observed in the AVCP protein. A spectroscopic study clearly showed that some ligands were bound to the PHCP protein. It is concluded that the dimeric PHCP from the thermophile is effectively stabilized through heme‐related and subunit–subunit interactions with conservation of the ligand‐binding ability.Brief SummaryWe report the X‐ray crystal structure of cytochrome c′ (PHCP) from thermophilic Hydrogenophilus thermoluteolus. The high thermal stability of PHCP was attributed to heme‐related and subunit–subunit interactions, which were confirmed by a mutagenesis study. The ligand‐binding ability of PHCP was examined by spectrophotometry. PHCP acquired the thermal stability with conservation of the ligand‐binding ability. This study furthers the understanding of the stability and function of cytochromes c.

One fold, two functions: cytochrome P460 and cytochrome c′-β from the methanotroph Methylococcus capsulatus (Bath)

“…The unfolding transitions are much broader for McP460 and McCP-β as is typical for β-sheet proteins in comparison to those of cyts cp-α. 31 , 32 We note for context that the optimum growth temperature of M. capsulatus (Bath) is 45 °C. 33 …”

Conferment of CO-Controlled Dimer–Monomer Transition Property to Thermostable Cytochrome c′ by Mutation in the Subunit–Subunit Interface

“…Wild-type PHCP and its variant plasmids were prepared as reported previously. 34 Phe11, Thr18, and Phe71 in the subunit-subunit interface were substituted to Thr (F11T), Phe (T18F), and Asp (F71D), respectively, which are the corresponding residues of AVCP. The T18F/F71D double and F11T/T18F/F71D triple variants were prepared by multistep mutagenesis using plasmids and primers of the single variants.…”

“…31 Thermophilic Hydrogenophilus thermoluteolus CP (PHCP; the same bacterium was previously named Pseudomonas hydrogenothermophila) exhibits a high similarity with AVCP in amino acid sequence and three-dimensional structure; 55% amino acid sequence identity, homo-dimeric structure with four-helix bundle subunits and Tyr16 at the heme sixth coordination site. 33,34 PHCP also binds exogenous ligands such as CO and NO, although PHCP is more stable than AVCP. 34 Three of the six residues responsible for the high stability of PHCP are located in the subunit-subunit interface: Phe11, Thr18, and Phe71 (Figure 1).…”

“…33,34 PHCP also binds exogenous ligands such as CO and NO, although PHCP is more stable than AVCP. 34 Three of the six residues responsible for the high stability of PHCP are located in the subunit-subunit interface: Phe11, Thr18, and Phe71 (Figure 1). 34 Phe11 forms a hydrophobic packing with Leu3 and Leu60 of the same subunit and Trp21 of the other subunit.…”

“…34 Three of the six residues responsible for the high stability of PHCP are located in the subunit-subunit interface: Phe11, Thr18, and Phe71 (Figure 1). 34 Phe11 forms a hydrophobic packing with Leu3 and Leu60 of the same subunit and Trp21 of the other subunit. The two Thr18(Oγ1) and two Ala14(O) residues of the respective subunits form an inter-subunit hydrogen-bonding network at the center of the subunit-subunit interface.…”