Open AccessEffect of the tyrosine 96 hydrogen bond on the inactivation of cytochrome P ‐450 cam induced by hydrostatic pressure
Author(s) -
PRIMO Carmelo,
HUI BON HOA Gaston,
DOUZOU Pierre,
SLIGAR Stephen
Publication year - 1990
Publication title -
european journal of biochemistry
Language(s) - English
Resource type - Journals
eISSN - 1432-1033
pISSN - 0014-2956
DOI - 10.1111/j.1432-1033.1990.tb19350.x
Subject(s) - cytochrome , chemistry , hydrostatic pressure , substrate (aquarium) , hydrogen bond , tyrosine , stereochemistry , mutant , heme , mutagenesis , hemeprotein , cytochrome c , enzyme , biochemistry , biology , organic chemistry , molecule , mitochondrion , ecology , physics , gene , thermodynamics
The effects of removal of the tyrosine 96 hydrogen bond on the stability and conformational events of cytochrome P ‐450 cam are presented in this communication. Hydrostatic pressure has been used as a tool to perturbe the structure leading to the formation of cytochrome P ‐420, an inactivated but soluble and undenatured form of the enzyme. We show that the spin transition of cytochrome P ‐450 cam , which is known to be influenced by hydrostatic pressure, is affected by this single mutation. The free energy of stabilisation of native substrate‐free cytochrome P ‐450 cam is not affected by the removal of the tyrosine 96 hydrogen bond via mutagenesis to phenylalanine, whereas the substrate‐bound protein shows a difference of 21 kJ/mol. These results, as well as an observed 110 ml/mol difference for the volume of the inactivation reaction between substrate‐bound native and mutant proteins, have been interpreted in terms of a more hydrated heme pocket for the site‐directed mutant at position 96 compared to the wild‐type protein where camphor is tightly bound via the tyrosine 96 hydrogen bond and water excluded from the active site.