Variable-temperature, variable-field magnetic circular dichroism spectroscopic study of the metal clusters in the Delta nifB and Delta nifH MoFe proteins of nitrogenase from Azotobacter vinelandii

摘要

Deletion of nifB results in the formation of a variant nitrogenase MoFe protein ( Delta nifB MoFe protein) that appears to contain two normal [ 8Fe-7S] P clusters. This protein can be reactivated to form the holo MoFe protein upon addition of isolated FeMo cofactor. In contrast, deletion of nifH results in a variant protein ( Delta nifH MoFe protein) that appears to contain FeS clusters different from the normal P cluster, presumably representing precursors of the normal P cluster. The Delta nifH MoFe protein is not reconstituted to the holo MoFe protein with isolated FeMo cofactor. The EPR and EXAFS spectroscopic properties of FeS clusters in the Delta nifH MoFe protein clearly differ from those of the normal P cluster found in the Delta nifB MoFe protein and suggest the presence of [ 4Fe-4S]-like clusters. To further characterize the metal cluster structures in the Delta nifH MoFe protein, a variable-temperature, variable-field magnetic circular dichroism ( VTVH-MCD) spectroscopic study has been undertaken on both the Delta nifB MoFe protein and the Delta nifH MoFe protein in both the dithionite-reduced and oxidized states. This study clearly shows that each half of the dithionite-reduced Delta nifH MoFe protein contains a [ 4Fe-4S](+) cluster paired with a diamagnetic [ 4Fe-4S]-like cluster. Upon oxidation, the VTVH-MCD spectrum of the Delta nifH MoFe protein reveals a paramagnetic, albeit EPR-silent system, suggesting an integer spin state. These results suggest that the Delta nifH MoFe protein contains a pair of neighboring, unusual [ 4Fe-4S]-like clusters, which are paramagnetic in their oxidized state.