The femoco-deficient MoFe protein produced by a nifH deletion strain of Azotobacter vinelandii shows unusual P-cluster features

Abstract

The His-tag MoFe protein expressed by the nifH deletion strain Azotobacter vinelandii DJ1165 (ΔnifH MoFe protein) was purified in large quantity. The α2β2 tetrameric ΔnifH MoFe protein is FeMoco-deficient based on metal analysis and the absence of the S = 3/2 EPR signal, which arises from the FeMo cofactor center in wild-type MoFe protein. The ΔnifH MoFe protein contains 18.6 mol Fe/mol and, upon reduction with dithionite, exhibits an unusually strong S = 1/2 EPR signal in the g ≈ 2 region. The indigo disulfonate-oxidized ΔnifH MoFe protein does not show features of the P2+ state of the P-cluster of the ΔnifB MoFe protein. The oxidized ΔnifH MoFe protein is able to form a specific complex with the Fe protein containing the [4Fe-4S]1+ cluster and facilitates the hydrolysis of MgATP within this complex. However, it is not able to accept electrons from the [4Fe-4S]1+ cluster of the Fe protein. Furthermore, the dithionite-reduced ΔnifH MoFe can be further reduced by Ti(III) citrate, which is quite unexpected. These unusual catalytic and spectroscopic properties might indicate the presence of a P-cluster precursor or a P-cluster trapped in an unusual conformation or oxidation state.