Hydrogenophilus thermoluteolus, Thermochromatium tepidum, and Allochromatium vinosum, which grow optimally at 52, 49, and 25 ºC, respectively, have homologous cytochromes c' (PHCP, TTCP, and AVCP, respectively) exhibiting at least 50% amino acid sequence identity. Here, the thermal stability of the recombinant TTCP protein was first confirmed to be between those of PHCP and AVCP. Structure comparison of the three proteins and a mutagenesis study on TTCP revealed that hydrogen bonds and hydrophobic interactions between the heme and amino acid residues were responsible for their stability differences. In addition, PHCP, TTCP, and AVCP, and their variants with altered stability similarly bound nitric oxide and carbon oxide, but not oxygen. Therefore, the thermal stability of TTCP together with PHCP and AVCP can be tuned through specific interactions around the heme without affecting their gas-binding function. These cytochromes c' will be useful as specific gas sensor proteins exhibiting a wide thermal stability range.

Cytochrome c′-β is a heme protein that belongs to the cytochrome P460 family and consists of homodimeric subunits with a predominantly antiparallel β-sheet fold. Here, the crystal structure of cytochrome c′-β from the thermophilic Thermus thermophilus (TTCP-β) is reported at 1.74 Å resolution. TTCP-β has a typical antiparallel β-sheet fold similar to that of cytochrome c′-β from the moderately thermophilic Methylococcus capsulatus (MCCP-β). The phenylalanine cap structure around the distal side of the heme is also similar in TTCP-β and MCCP-β, indicating that both proteins similarly bind nitric oxide and carbon monoxide, as observed spectroscopically. Notably, TTCP-β exhibits a denaturation temperature of 117°C, which is higher than that of MCCP-β. Mutational analysis reveals that the increased homodimeric interface area of TTCP-β contributes to its high thermal stability. Furthermore, 14 proline residues, which are mostly located in the TTCP-β loop regions, possibly contribute to the rigid loop structure compared with MCCP-β, which has only six proline residues. These findings, together with those from phylogenetic analysis, suggest that the structures of Thermus cytochromes c′-β, including TTCP-β, are optimized for function under the high-temperature conditions in which the source organisms live.