Instantaneous, stoichiometric generation of powerfully reducing states of protein active sites using Eu(II) and polyaminocarboxylate ligands.

Addition of an equivalent of a polyaminocarboxylate ligand (L) to a solution of a redox protein and the aqua Eu2+ ion results in the instantaneous in situ generation of a very powerful reductant Eu(II)-L that can rapidly drive an electron stoichiometrically onto a redox centre having an extremely negative reduction potential (lower than -1 V): this is exemplified by straightforward generation of the super-reduced state of the Fe-protein of nitrogenase.

Mechanisms of redox-coupled proton transfer in proteins: role of the proximal proline in reactions of the [3Fe-4S] cluster in Azotobacter vinelandii ferredoxin I.

The 7Fe ferredoxin from Azotobacter vinelandii (AvFdI) contains a [3Fe-4S](+/0) cluster that binds a single proton in its reduced level. Although the cluster is buried, and therefore inaccessible to solvent, proton transfer from solvent to the cluster is fast. The kinetics and energetics of the coupled electron-proton transfer reaction at the cluster have been analyzed in detail by protein-film voltammetry, to reveal that proton transfer is mediated by the mobile carboxylate of an adjacent surface residue, aspartate-15, the pK of which is sensitive to the charge on the cluster.

Direct assessment of the reduction potential of the [4Fe-4S](1+/0) couple of the Fe protein from Azotobacter vinelandii.

Recently, it has been demonstrated that the [4Fe-4S] cluster of the Fe protein of nitrogenase from Azotobacter vinelandii can be reduced to an unprecedented all-ferrous state. In this work, the reduction potential for the formation of the all-ferrous state is measured by the reactions of the reduced and oxidized Fe protein with a variety of chemical redox active agents, and by mediated spectroelectrochemical titration. Redox titrations obtain a potential ca.

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

The His-tag MoFe protein expressed by the nifH deletion strain Azotobacter vinelandii DJ1165 (Delta(nifH) MoFe protein) was purified in large quantity. The alpha(2)beta(2) tetrameric Delta(nifH) 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 Delta(nifH) MoFe protein contains 18.

Structure of a cofactor-deficient nitrogenase MoFe protein.

One of the most complex biosynthetic processes in metallobiochemistry is the assembly of nitrogenase, the key enzyme in biological nitrogen fixation. We describe here the crystal structure of an iron-molybdenum cofactor-deficient form of the nitrogenase MoFe protein, into which the cofactor is inserted in the final step of MoFe protein assembly. The MoFe protein folds as a heterotetramer containing two copies each of the homologous alpha and beta subunits.

Crystal structures of ferredoxin variants exhibiting large changes in [Fe-S] reduction potential.

Elucidating how proteins control the reduction potentials (E0') of [Fe--S] clusters is a longstanding fundamental problem in bioinorganic chemistry. Two site-directed variants of Azotobacter vinelandii ferredoxin I (FdI) that show large shifts in [Fe--S] cluster E0' (100--200 mV versus standard hydrogen electrode (SHE)) have been characterized. High resolution X-ray structures of F2H and F25H variants in their oxidized forms, and circular dichroism (CD) and electron paramagnetic resonance (EPR) of the reduced forms indicate that the overall structure is not affected by the mutations and reveal that there is no increase in solvent accessibility nor any reorientation of backbone amide dipoles or NH--S bonds.

Azotobacter vinelandii ferredoxin I: a sequence and structure comparison approach to alteration of [4Fe-4S]2+/+ reduction potential.

The reduction potential (E(0)') of the [4Fe-4S](2+/+) cluster of Azotobacter vinelandii ferredoxin I (AvFdI) and related ferredoxins is approximately 200 mV more negative than the corresponding clusters of Peptostreptococcus asaccharolyticus ferredoxin and related ferredoxins. Previous studies have shown that these differences in E(0)' do not result from the presence or absence of negatively charged surface residues or in differences in the types of hydrophobic residues found close to the [4Fe-4S](2+/+) clusters. Recently, a third, quite distinct class of ferredoxins (represented by the structurally characterized Chromatium vinosum ferredoxin) was shown to have a [4Fe-4S](2+/+) cluster with a very negative E(0)' similar to that of AvFdI.