Enzymatic activation of cytochrome P-450(cam)

Abstract

Cytochrome P-450$\sb{\rm cam}$, a camphor monoxygenase from Pseudomonas putida, has served as a model system for the entire family of the P-450s in exploring structure-function relationships, molecular recognition, substrate specificity, and oxygen activation. There are several universal features of the P-450s that have been addressed in this thesis work. The active site is centered around the heme prosthetic group, which is primarily responsible for the redox activity of the enzyme. Iron chlorin groups, which are partially saturated analogues of protoporphyrin IX, were substituted for the native heme in cytochrome P-450$\sb{\rm cam}$ and also rat liver cytochrome $b\sb5$, which served as a useful model for the more difficult reconstitution and subsequent characterization of the P-450 enzyme. Optical and electron paramagnetic resonance spectroscopies were used to study the physical properties of the reconstituted proteins. In addition reduction potentials were measured and the ability of the chlorin-substituted proteins to carry out native and redox activity was investigated.

A second project focused on the inactivated form of the protein, cytochrome P-420. This transition is common to all of the P-450s and has been widely speculated to be linked to a modification of the unusual axial fifth ligand, a cysteine-derived thiolate group. High pressure-induced species of cytochrome P-420$\sb{\rm cam}$ were employed to evaluate the alterations to the inactivated enzyme. It was found that the lack of activity was due at least in part to an inability of the protein to bind camphor, which results in an uncoupling of the thermodynamic regulation mechanism of electron transfer. In addition, chemical and physical data supported a retention of the thiolate ligand to the heme in the ferric state of cytochrome P-420$\sb{\rm cam}$. The axial ligand bond appeared to be either stretched or broken upon reduction.

The final portion of this dissertation explores the oxygen binding pocket using a mutant cytochrome P-450$\sb{\rm cam}$, which contains an alanine in replacement of a highly conserved threonine residue. Characterization of the mutant protein not only showed that this amino acid stabilized the dioxygen intermediates of cytochrome P-450$\sb{\rm cam}$ against autoxidation, but it also implicated the threonine residue as playing an important role in oxygen activation.