Five amino acid residues responsible for the high stability of Hydrogenobacter thermophilus cytochrome c552 -: Reciprocal mutation analysis

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/ 178V) showed the same stability against guanidine hydrochloride denaturation as that of PA C-511, suggesting that the five residues in HT C-552 necessarily and sufficiently contribute to the overall stability. In the three HT C551 variants carrying mutations in each of the three regions, the Y34F/Y43E mutations resulted in the greatest destabilization, by -13.3 kJ mol(-1), followed by A7F/M13V (-3.3 W mol(-1)) and then 178V (-1.5 W mol(-1)). The order of destabilization in HT C-552 was the same as that of stabilization in PA C-551 with reverse mutations such as F34Y/ E43Y, F7A/V13WL and V78I (13.4, 10.3, and 0.3 W mol(-1), 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 nmino acid contribution to the overall stability.