The effects of removal of the tyrosine 96 hydrogen bond on the stability and conformational events of cytochrome P‐450cam 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‐450cam, 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‐450cam 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.
|Original language||English (US)|
|Number of pages||4|
|Journal||European Journal of Biochemistry|
|State||Published - Oct 1990|
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