ABSTRACT
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.
Subject(s)
Egtazic Acid/chemistry , Europium/chemistry , Nitrogenase/chemistry , Pentetic Acid/chemistry , Azotobacter vinelandii/enzymology , Binding Sites , Egtazic Acid/analogs & derivatives , Electrochemistry , Ligands , Nonheme Iron Proteins/chemistry , Organometallic Compounds/chemistry , Oxidation-Reduction , Pentetic Acid/analogs & derivativesABSTRACT
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. The structures, combined with detailed investigation of the variation of E0' with pH and temperature, show that the largest increases in E0' result from the introduction of positive charge due to protonation of the introduced His residues. The smaller (50--100 mV) increases observed for the neutral form are proposed to occur by directing a Hdelta+--Ndelta- dipole toward the reduced form of the cluster.
Subject(s)
Azotobacter vinelandii/chemistry , Ferredoxins/chemistry , Ferredoxins/metabolism , Mutation/genetics , Bacterial Proteins/chemistry , Bacterial Proteins/genetics , Bacterial Proteins/metabolism , Circular Dichroism , Crystallography, X-Ray , Electrochemistry , Electron Spin Resonance Spectroscopy , Ferredoxins/genetics , Hydrogen/metabolism , Hydrogen Bonding , Hydrogen-Ion Concentration , Models, Molecular , Oxidation-Reduction , Protein Conformation , Protons , Structure-Activity Relationship , TemperatureABSTRACT
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. The observation that the sequences and structures surrounding the very negative E(0)' clusters in quite dissimilar proteins were almost identical inspired the construction of three additional mutations in the region of the [4Fe-4S](2+/+) cluster of AvFdI. The three mutations, V19E, P47S, and L44S, that incorporated residues found in the higher E(0)' P. asaccharolyticus ferredoxin all led to increases in E(0)' for a total of 130 mV with a 94-mV increase in the case of L44S. The results are interpreted in terms of x-ray structures of the FdI variants and show that the major determinant for the large increase in L44S is the introduction of an OH-S bond between the introduced Ser side chain and the Sgamma atom of Cys ligand 42 and an accompanying movement of water.
