Effects of heme on the thermal stability of mesophilic and thermophilic cytochromes c : Comparison between experimental and theoretical results

We have recently proposed a measure of the thermal stability of a protein: the water-entropy gain at 25°C upon folding normalized by the number of residues, which is calculated using a hybrid of the angle-dependent integral equation theory combined with the multipolar water model and the morphometric approach. A protein with a larger value of the measure is thermally more stable. Here we extend the study to analyses on the effects of heme on the thermal stability of four cytochromes c (PA c 551 , PH c 552 , HT c 552 , and AA c 555 ) whose denaturation temperatures are considerably different from one another despite that they share significantly high sequence homology and similar three-dimensional folds. The major conclusions are as follows. For all the four cytochromes c , the thermal stability is largely enhanced by the heme binding in terms of the water entropy. For the holo states, the measure is the largest for AA c 555 . However, AA c 555 has the lowest packing efficiency of heme and the apo polypeptide with hololike structure, which is unfavorable for the water entropy. The highest stability of AA c 555 is ascribed primarily to the highest efficiency of side-chain packing of the apo polypeptide itself. We argue for all the four cytochromes c that due to covalent heme linkages, the number of accessible conformations of the denatured state is decreased by the steric hindrance of heme, and the conformational-entropy loss upon folding becomes smaller, leading to an enhancement of the thermal stability. As for the apo state modeled as the native structure whose heme is removed, AA c 555 has a much larger value of the measure than the other three. Overall, the theoretical results are quite consistent with the experimental observations (e.g., at 25°C the α-helix content of the apo state of AA c 555 is almost equal to that of the holo state while almost all helices are collapsed in the apo states of PA c 551 , PH c 552 , and HT c 552 ).