The most recognized activity of P450cam is the oxidation of the unactivated C-H bond at C-5 of D (+)-camphor to an alcohol moiety. This hydroxylation reaction has few counterparts in chemical synthesis; hence, the application of cytochrome P450cam for industrial purposes has practical potential. P450¬cam requires a carefully orchestrated reaction cycle, which includes two electron transfer partners: putidaredoxin (Pdx) and putidaredoxin reductase (Pdr). Studies have shown that Pdx plays an essential role in electron transfer and in controlling key steps in the catalytic cycle. In this thesis, the multiple-component system dependency of P450cam was addressed by chemically linking P450cam to Pdx. The linker bridging the two proteins was either a 4-carbon or 7-carbon saturated alkyl group with a functional bipyridyl anchor. This linker was chemically attached to a cysteine residue on the surface of P450cam. The histidine groups on the C-terminus of Pdx and the bipyridyl anchor on P450cam tethered the two proteins together via a coordination bridge with a metal (Ni2+ or Ru3+). The new entities showed reasonable stability and reactivity, provided a framework that improved the stability of Pdx, and did not exhibit uncoupling. In order to study the product profile of cytochrome P450cam¬ upon catalysis, a set of standards was required. The second objective of my thesis was the synthesis and characterization of the different compounds metabolized by P450cam, namely, 5-ketocamphor, 5-exo-hydroxycamphor, 5-exo-hydroxyborneol and 5-exo-hydroxyisoborneol. They were useful as standards to follow the conversion pathways of bicyclic metabolites by Pseudomonas putida’s enzymes in vivo and the activity of the linked system in vitro. In the last part of this thesis, the newly discovered actvities of cytochrome P450cam were investigated. Under high oxygen, the P450cam system over-oxidizes D (+)-camphor to (+)-5-ketocamphor by a double hydroxylation at C-5 on the bicyclic skeleton. Moreover, under low oxygen and reducing conditions, the P450cam system catalyzes the reduction of the 2-keto group of D (+)-camphor or (+)-5-exo-hydroxycamphor to (+)-borneol or (+)-5-exo-hydroxyborneol respectively. Subsequently, it was shown that Pdr enhances the reduction reaction under anoxic conditions and that the hydrogen that is installed at C-2 comes from water.
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