Misa Masanari

Conferment of folding ability to a naturally unfolded apocytochrome c through introduction of hydrophobic amino acid residues.

Hyperthermophilic Aquifex aeolicus cytochrome c(555) (AA c(555)) exceptionally folds even in the apo state, unlike general cytochromes c including mesophilic Pseudomonas aeruginosa cytochrome c(551) (PA c(551)), which is structurally homologous to AA c(555) in the holo state. Here we hypothesized that the exceptional apo AA c(555) folding can be attributed to nine hydrophobic amino acid residues and proved this using a PA c(551) variant (denoted as PA-nh) carrying the nine hydrophobic residues at structurally corresponding positions. Circular dichroism experiments showed that the apo PA-nh variant became folded, unlike the wild-type apo PA c(551), and exhibited much higher stability than the wild type. Another difference between the holo forms of AA c(555) and PA c(551) is the existence of an extra helix in the former. Introduction of the amino acid residues forming the extra helix of AA c(555) into the PA-nh variant did not significantly affect its folding ability in the apo state. Therefore, the nine hydrophobic residues introduced into the apo PA-nh variant were enough to confer the folding ability. PA c(551) represents the first example of the conversion of an intrinsically unfolded apocytochrome c into an autonomously folded one, which was revealed by means of a protein engineering method without heme. Although heme is generally considered to be a trigger of apocytochrome c folding, the present results demonstrate a new heme-independent folding mechanism.

High Thermal Stability and Unique Trimer Formation of Cytochrome c′ from Thermophilic Hydrogenophilus thermoluteolus

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.

Correlation between the optimal growth pressures of four Shewanella species and the stabilities of their cytochromes c 5

Shewanella species live widely in deep-sea and shallow-water areas, and thus grow piezophilically and piezosensitively. Piezophilic and psychrophilic Shewanella benthica cytochrome c5 (SB cytc5) was the most stable against guanidine hydrochloride (GdnHCl) and thermal denaturation, followed by less piezophilic but still psychrophilic Shewanella violacea cytochrome c5 (SV cytc5). These two were followed, as to stability level, by piezosensitive and mesophilic Shewanella amazonensis cytochrome c5 (SA cytc5), and piezosensitive and psychrophilic Shewanella livingstonensis cytochrome c5 (SL cytc5). The midpoint GdnHCl concentrations of SB cytc5, SV cytc5, SL cytc5, and SA cytc5 correlated with the optimal growth pressures of the species, the correlation coefficient value being 0.93. A similar trend was observed for thermal denaturation. Therefore, the stability of each cytochrome c5 is related directly to its host’s optimal growth pressure. Phylogenetic analysis indicated that Lys-37, Ala-41, and Leu-50 conserved in piezosensitive SL cytc5 and SA cytc5 are ancestors of the corresponding residues in piezophilic SB cytc5 and SV cytc5, Gln, Thr, and Lys, respectively, which might have been introduced during evolution on adaption to environmental pressure. The monomeric Shewanella cytochromes c5 are suitable tools for examining protein stability with regard to the optimal growth pressures of the source species.

Thermal Stability of Cytochrome c 5 of Pressure-Sensitive Shewanella livingstonensis

Cytochrome c 5 of pressure-sensitive Shewanella livingstonensis (SL cytc 5) exhibits lower thermal stability than a highly homologous counterpart of pressure-tolerant Shewanella violacea. This stability difference is due to an enthalpic effect that can be attributed to the amino acid residue at position 50 (Leu or Lys). These cytc 5 proteins are appropriate materials for understanding the protein stability mechanism.

Comparative study on stabilization mechanism of monomeric cytochrome c5 from deep-sea piezophilic Shewanella violacea

Monomeric cytochrome c5 from deep-sea piezophilic Shewanella violacea (SVcytc5) was stable against heat and denaturant compared with the homologous protein from shallow-sea piezo-sensitive Shewanella livingstonensis (SLcytc5). Here, the SVcytc5 crystal structure revealed that the Lys-50 side chain on the flexible loop formed a hydrogen bond with heme whereas that of corresponding hydrophobic Leu-50 could not form such a bond in SLcytc5, which appeared to be one of possible factors responsible for the difference in stability between the two proteins. This structural insight was confirmed by a reciprocal mutagenesis study on the thermal stability of these two proteins. As SVcytc5 was isolated from a deep-sea piezophilic bacterium, the present comparative study indicates that adaptation of monomeric SVcytc5 to high pressure environments results in stabilization against heat. Graphical abstract Structure of Shewanella violaceacy to chrome c5 revealed that Lys-50 on a flexible loop was responsible for its stability.

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

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.

Thermal stability of cytochrome c' from mesophilic Shewanella amazonensis.

Cytochrome c′ (SACP) from mesophilic Shewanella amazonensis, growing optimally at 37 °C, was thermally more stable than cytochrome c′ (AVCP) from mesophilic Allochromatium vinosum, growing optimally at 25 °C. In contrast, SACP was less stable than cytochrome c′ (PHCP) from thermophilic Hydrogenophilus thermoluteolus, growing optimally at 52 °C. Although only 28% of the SACP amino acid sequence was identical to those of AVCP and PHCP, the latter two being 55% identical, the overall main chain structures of the three cytochromes c′ were similar, and SACP exhibited thermal stability intermediate between those of AVCP and PHCP. For these three proteins, the higher the stability is, the lesser the number of Gly residues in the putative α-helical regions is. Cytochromes c′ including the present three are suitable for examining the protein stabilization mechanisms, because they are structurally similar and available from environments with a wide range of temperatures. Cytochrome c′ from S. amazonensis, growing at 37 °C, exhibited stability intermediate between counterparts from A. vinosum and H. thermoluteolus, growing at 25 and 52 °C, respectively.

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

Apo-cytochomes c without heme are usually unstructured. Here we showed that apo-form of thermophilic Hydrogenophilus thermoluteolus cytochrome c’ (PHCP) was a monomeric protein with high helix content. Apo-PHCP was thermally stable, possibly due to the hydrophobic residues and ion pairs. PHCP is the first example of a structured apo-cytochrome c’, which will expand our view of hemoprotein structure formation.

Oxidative phosphorylation in a thermophilic, facultative chemoautotroph, Hydrogenophilus thermoluteolus, living prevalently in geothermal niches

Hydrogenophilus is a thermophilic, facultative chemoautotroph, which lives prevalently in high temperature geothermal niches. Despite the environmental distribution, little is known about its oxidative phosphorylation. Here, we show that inverted membrane vesicles derived from Hydrogenophilus thermoluteolus cells autotrophically cultivated with H2 formed a proton gradient on the addition of succinate, dl-lactate, and NADH, and exhibited oxidation activity toward these three organic compounds. These indicate the capability of mixotrophic growth of this bacterium. Biochemical analysis demonstrated that the same vesicles contained an F-type ATP synthase. The F1 sector of the ATP synthase purified from H. thermoluteolus membranes exhibited optimal ATPase activity at 65°C. Transformed Escherichia coli membranes expressing H. thermoluteolus F-type ATP synthase exhibited the same temperature optimum for the ATPase. These findings shed light on H. thermoluteolus oxidative phosphorylation from the aspects of membrane bioenergetics and ATPase biochemistry, which must be fundamental and advantageous in the biogeochemical cycles occurred in the high temperature geothermal niches.