Five Amino Acid Residues Responsible for the High Stability of Hydrogenobacter thermophilus Cytochrome c552

Abstract

Five amino acid residues responsible for extreme stability have been identified in cytochrome c 552 (HT c 552 ) from a thermophilic bacterium, Hydrogenobacter thermophilus. The five residues, which are spatially distributed in three regions of HT c 552 , were replaced with the corresponding residues in the homologous but less stable cytochrome c 551 (PA c 551 ) from Pseudomonas aeruginosa. The quintuple HT c 552 variant (A7F/M13V/Y34F/Y43E/ I78V) showed the same stability against guanidine hydrochloride denaturation as that of PA c 551 , suggesting that the five residues in HT c 552 necessarily and sufficiently contribute to the overall stability. In the three HT c 552 variants carrying mutations in each of the three regions, the Y34F/Y43E mutations resulted in the greatest destabilization, by ؊13.3 kJ mol , respectively). The results of guanidine hydrochloride denaturation were consistent with those of thermal denaturation for the same variants. The present study established a method for reciprocal mutation analysis. The effects of side-chain contacts were experimentally evaluated by swapping the residues between the two homologous proteins that differ in stability. A comparative study of the two proteins was a useful tool for assessing the amino acid contribution to the overall stability.Proteins from thermophilic bacteria usually exhibit enhanced stability against temperature or denaturants compared with the homologues from mesophiles (1, 2). Sequence comparison and rationally designed mutations of thermophilic and mesophilic proteins provide several lines of information on protein stability. In particular, investigation of the relationship between three-dimensional structure and thermodynamic parameters on protein unfolding provides detailed information on factors contributing to the stability.A thermophilic hydrogen-oxidizing Gram-negative bacterium, Hydrogenobacter thermophilus, which grows optimally at 72°C, produces a periplasmic Class I cytochrome c 552 (HT c 552 ) 1 (3). This bacterial cytochrome c has greatly contributed to the understanding of protein stability through pairwise comparison with homologous cytochrome c 551 (PA c 551 ) from a mesophilic bacterium, Pseudomonas aeruginosa, which grows at 37°C (4). These two proteins exhibit 56% sequence identity and have almost the same backbone conformations, but HT c 552 is much more stable than PA c 551 (5-9).On precise structural comparison between HT c 552 and PA c 551 , we predicted that five amino acid residues spatially located in three regions were responsible for the higher stability of HT c 552 (6). These residues were then introduced at the corresponding positions in PA c 551 (7-9). The single mutation Val-78 to Ile (V78I) and two double mutations Phe-7 to Ala/ Val-13 to Met (F7A/V13M) and Phe-34 to Tyr/Glu-43 to Tyr (F34Y/E43Y) in the corresponding three regions of PA c 551 resulted in enhanced protein stability in an additive manner. Although the five residues were proved to be effective for stability, e.g. the denaturation temperature was e...

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

“…These values are significantly smaller than those obtained in our previous GdnHCl-induced denaturation experiments on the oxidized forms, 6,9,10) confirming that GdnSCN is a stronger denaturant than GdnHCl, as reported previously.…”

“…The HT c 552 , PH c 552 , and PA c 551 wild-type proteins, and the reciprocal variants, HT c 552 I78V, PH c 552 V78I, and PA c 551 V78I, were prepared by the method previously described. 6,9,10) We adopted the residue numbering system used for PA c 551 for clarity. The proteins were overexpressed with co-transformed pEC86 carrying the ccmABCDEFGH genes in Escherichia coli JCB387 cells and isolated from periplasmic extracts.…”

“…The proteins were purified by HiTrap Q and Hi-Trap SP column chromatography (GE Healthcare, Piscataway, NJ) with a sodium chloride concentration gradient (0-500 mM), as described previously. 6,9,10) Finally, gel-filtration chromatography was performed on a Superdex 75 column (GE Healthcare, Piscataway, NJ) equilibrated and eluted with 25 mM sodium acetate buffer, pH 5.0.…”

Effects of Cysteine Introduction into Three Homologous Cytochromesc

“…The HT c 552 , PH c 552 , and PA c 551 wild-type proteins, and the variants, HT c 552 S78C, PH c 552 T77C, and PA c 551 S80C, were prepared by a method previously described, 6,8,9) with minor modifications. The proteins were overexpressed with co-transformed pEC86 carrying the ccmABCDEFGH genes in Escherichia coli JCB387 cells and isolated from the periplasmic extracts.…”

Unexpected Elevated Production ofAquifex aeolicusCytochromec555inEscherichia coliCells Lacking Disulfide Oxidoreductases

“…First we examined whether E. coli strain RI89 (MC1000 phoR Áara-714 leu þ ), 5) used as the ''wild type'' would produce holo forms of cytochrome c in the periplasm. The cytochromes c examined in this study were Pseudomonas aeruginosa cytochrome c 551 (PA c 551 ), 6) Hydrogenophilus thermoluteolus cytochrome c 552 (PH c 552 ), 7) Hydrogenobacter thermophilus cytochrome c 552 (HT c 552 ), 8) A. aeolicus cytochrome c 555 (AA c 555 ), 9) and human mitochondrial and Arabidopsis thaliana chloroplast cytochromes c. 10,11) To target the cytochrome c polypeptides to the E. coli periplasm, the gene for the signal sequence of PA c 551 was fused in-frame with those encoding the mature regions of other cytochromes c by the method of Zhang et al…”