title: Effect of the tyrosine 96 hydrogen bond on the inactivation of cytochrome P-450cam induced by hydrostatic pressure.
abstract: 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.
authors.affiliation: Unité 310, Institut National de la Santé et de la Recherche Médicale-Institut National de la Recherche Agrónomique, Paris, France.
authors.forename: C
authors.initials: C
authors.lastname: Di Primo
authors.forename: G
authors.initials: G
authors.lastname: Hui Bon Hoa
authors.forename: P
authors.initials: P
authors.lastname: Douzou
authors.forename: S
authors.initials: S
authors.lastname: Sligar
mesh_terms: D002164:Camphor; D003577:Cytochrome P-450 Enzyme System / Q000737:chemistry* / Q000235:genetics; D003580:Cytochromes / Q000737:chemistry* / Q000235:genetics; D006860:Hydrogen Bonding; D006874:Hydrostatic Pressure; D016297:Mutagenesis, Site-Directed; D010649:Phenylalanine / Q000737:chemistry; D011549:Pseudomonas / Q000201:enzymology; D013057:Spectrum Analysis; D014443:Tyrosine / Q000737:chemistry* / Q000235:genetics
chemical_list: D003580:Cytochromes; D014443:Tyrosine; D010649:Phenylalanine; D002164:Camphor; C013464:cytochrome P420; D003577:Cytochrome P-450 Enzyme System