| Cytochrome P450s, a kind of heme-containing superfamily found in animals, plants and microorganisms, catalyze biosynthesis of endogenous compounds and metabolism of xenobiotics. They are widely used in chemistry, pharmacy, biology and bioengineering for their powerful catalysis abilities and variable reaction types. P450s catalyse their substrates when receiving the electrons from other proteins, which constitute the P450electron transport systems with P450s. According to the differences of electron transport proteins, the systems are divided into ten types and the class I and II are the most prevalent types. Bacterium usually have the class I electron transport system consisting of a ferredoxin reductase, a ferredoxin and a P450protein.Rhodopseudomonas palustris HaA2has highly adaptability to endure high concentrated organic wastewater and catabiosis toxic compounds such as phenol and cyanogen. Two class I P450electron transport systems, HaPuR-HaPux-CYP199A4and RPB3630(HaPuxC)-RPB361(ferredoxin reductase is not identified yet), were found there. Here, the expression, purification, crystallization and structure determination of two ferredoxins (HaPuxC and HaPux) were reported. HaPuxC belongs to3Fe-4S cluster ferredoxin, which consists of two helixs, two antiparallel β sheets and five turns. Compared with other ferredoxins, the β sheets (β2and β3) and the turn between β sheet and helix2are longer. As for the CXXXXXC(X)nCP motif, X is Histidine in this structure, which forms hydrogen bonds with the conserved Glu61residing in Fe-S cluster binding loop. The Glu61was expected to play important role in P450recognition, while the role of His is still further studied. The HaPux is2Fe-2S cluster ferredoxin protein, with four conserved cysteine residues Cys39ã€Cys45ã€Cys48and Cys86in each molecule, which are ligands to the metal cluster. Compared with other2Fe-2S type ferredoxins, there was no redundant cysteine residues in HaPux. The conserved histidine behind the third cysteine, involving in the formation of intramolecular hydrogen bond network, is vital for the molecular configuration maintaining. The solving of HaPuxC and HaPux structures replenished the type I electron transport chain and gave clues for the research of ferredoxin reductase. Meanwhile, comparing the surface charges of the loop which binding Fe-S cluster (the domain where ferredoxin recognizes and interacts with P450) between HaPuxC and HaPux, we verify the mechanism that different ferredoxins can recognize and interacte with specific P450s by the interaction of surface charges.The second part of this thesis is the structure determination of some CYP1O2A1(BM3) mutations from Bacillus subtilis. Different BM3mutations can bind and catalyse specific substrates. They can also be used for mimic human P450proteins catabiosising some clinical drugs. Three mutants were selected for structure solving and drug soaking. By using structure alignments, we found the overall structures were similar with the native structure. While the intramolecular hydrogen bonds and the sizes of substrate access channel were changed once the amino acid residues residing in active site or passage pocket were mutated, which resulted in the substrate specificity variation. While the detailed interactions between the mutant and the non-native substrates still rely on the solving of complex with the drugs. |
