Studies of Electron Transport from Flavodoxin Reductase to NrdI in Bacillus cereus

Abstract

Ribonucleotide reductase (RNR) catalyses the reduction of the four ribonucleotides to their corresponding deoxyribonucleotides needed for synthesis and repair of DNA. RNR is found in the genome of all living organisms, indicating that this enzyme is an essential part of the fundamentals of life. The RNRs explored to date are divided into three different main classes; Ia-c, II and III. The cofactor of class Ib RNRs was recently shown to be a imanganese cluster. Further that the flavoprotein NrdI is essential for the generation of the dimanganese-tyrosyl radical, the active form of the cofactor. An active cofactor is, in turn, essential for generation of a protein radical in the catalytic site, which initiates substrate turnover. The function of NrdI in class Ib RNR is, however, not fully understood. Two flavodoxin reductases (FldRs) have been located in the genome of Bacillus cereus (B. cereus), and considered as potential reducers of NrdI by transferring electrons from NADPH to NrdI. Investigations in their ability to interact with and reduce NrdI, would enhance the knowledge about the function of NrdI. This master project has focused on the two FldRs found in B. cereus. One of them was cloned, and both were expressed and purified. One of the FldRs is expressed primary as apo protein, and work was performed to reconstitute cofactor-containing protein from apo-FldR and flavin. Initial protein crystals were obtained for both proteins, but no complete datasets could be collected. The ability of each FldR to reduce NrdI and two other flavodoxins from B. cereus was tested. The result suggests that the redox partners of both FldRs are other than NrdI. Initial kinetic measurements were performed with 2,6-dichlorophenolindophenol (DCPIP) as the electron acceptor, resulting in a 200-fold difference in the Km values of the respective FldRs. Their ability to reduce cytochrome c (Cytc) was also tested. In sum, the experiments with the two-electron acceptor DCPIP and the one-electron acceptor Cytc indicated that one FldR is the most effective two-electron donor, and the other FldR is the most effective one-electron donor.