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...

Cys-59 and Cys-62, forming a disulfide bond in the four-residue loop of Shewanella violacea cytochrome c (5) (SV cytc (5)), contribute to protein stability but not to redox function. These Cys residues were substituted with Ala in SV cytc (5), and the structural and functional properties of the resulting C59A/C62A variant were determined and compared with those of the wild-type. The variant had similar features to those of the wild-type in absorption, circular dichroic, and paramagnetic (1)H NMR spectra. In addition, the redox potentials of the wild-type and variant were essentially the same, indicating that removal of the disulfide bond from SV cytc (5) does not affect the redox function generated in the vicinity of heme. However, calorimetric analysis of the wild-type and variant showed that the mutations caused a drastic decrease in the protein stability through enthalpy, but not entropy. Four residues are encompassed by the SV cytc (5) disulfide bond, which is the shortest one that has been proved to affect protein stability. The protein stability of SV cytc (5) can be controlled without changing the redox function, providing a new strategy for regulating the stability and function of cytochrome c.

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Cytochrome c(552) (PH c(552)) from moderately thermophilic Hydrogenophilus thermoluteolus exhibits stability intermediate between those of cytochrome c(552) (HT c(552)) from thermophilic Hydrogenobacter thermophilus and cytochrome c(551) (PA c(551)) from mesophilic Pseudomonas aeruginosa. To understand the mechanism of stabilization of PH c(552), we introduced mutations into PH c(552) at five sites, which, in HT c(552), are occupied by the amino acids responsible for stability higher than the less stable PA c(551). When PH c(552) Val-78 was mutated to Ile, as found in HT c(552), the resulting variant showed increased stability. Mutation of Ala-7, Met-13, and Tyr-34 to the corresponding residues in PA c(551) (Phe, Val, and Phe, respectively) resulted in destabilization. We also found that PH c(552) Lys-43 contributed to stability through the formation of an attractive electrostatic interaction with Asp-39. These results suggest that the intermediate stability of PH c(552) is due to the amino acids at these five sites.

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The chemical denaturation of Pseudomonas aeruginosa cytochrome c(551) variants was examined at pH 5.0 and 3.6. All variants were stabilized at both pHs compared with the wild-type. Remarkably, the variants carrying the F34Y and/or E43Y mutations were more stabilized than those having the F7A/V13M or V78I ones at pH 5.0 compared with at pH 3.6 by ~3.0-4.6 kJ/mol. Structural analyses predicted that the side chains of introduced Tyr-34 and Tyr-43 become hydrogen donors for the hydrogen bond formation with heme 17-propionate at pH 5.0, but less efficiently at pH 3.6, because the propionate is deprotonated at the higher pH. Our results provide an insight into a stabilization strategy for heme proteins involving variation of the heme electronic state and introduction of appropriate mutations.

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