Characterization of the novel electron transport chain P450cin, cindoxin, and cindoxin reductase

P450s have been divided into two main classes for the past 20 years, based upon systems of electron transport. Electron transport to P450cin was recently discovered to be an intermediate system, with characteristics of both classes, as well as a strong homology to the human cytochrome P450 reductase (CPRFMN). We propose that the electron transport partners of P450cin, cindoxin (CDX) and cindoxin reductase (CDR), would make an excellent bacterial model for the human reductase. Therefore, this work focuses on the characterization of P450cin and its two electron transport partners via spectroscopy, fluorescence, stop-flow kinetics, potentiometry, x-ray crystallography, and NMR. Expression and purification of CDR continues to be problematic, and no pure protein has yet been isolated. The purification and expression of CDX has been optimized in this work in both complex and minimal media. The kinetic characterization, as well as binding specificity was examined between P450cin and CDX and shown to be comparable to other known electron transport systems. The potentials of CDX were shown to be very similar to CPRFMN in contrast to those of the current model P450BM3. Crystals of CDX formed readily in high salt conditions, but were thin and were unable to be indexed due to multiple lattices. An energy minimized homology model for CDX was constructed based on the crystal structure of CPRFMN which displayed the characteristic fold of flavoprotiens. From that model a binding model of CDX-P450cin was built based on the crystal structure of P450BM3. These two models guided mutations for crystallization and kinetics studies. The binding surface was investigated through NMR, through the use of pseudocontact shifts as CDX came close to P450cin heme iron. The natural fluorescence of the cofactor FMN was used to optimize temperature and salt conditions to stabilize CDX for NMR data collection. Although many spectra have been collected, the sequence assignment of CDX has not yet been completed. This work represents the groundwork for the study of a new system of electron transport to P450.