Electrochemical and structural characterization of Azotobacter vinelandii flavodoxin II

Abstract

Azotobacter vinelandii flavodoxin II serves as a physiological reductant of nitrogenase, the enzyme system mediating biological nitrogen fixation. Wildtype A. vinelandii flavodoxin II was electrochemically and crystallographically characterized to better understand the molecular basis for this functional role. The redox properties were monitored on surfactant‐modified basal plane graphite electrodes, with two distinct redox couples measured by cyclic voltammetry corresponding to reduction potentials of −483 ± 1 mV and −187 ± 9 mV (vs. NHE) in 50 mM potassium phosphate, 150 mM NaCl, pH 7.5. These redox potentials were assigned as the semiquinone/hydroquinone couple and the quinone/semiquinone couple, respectively. This study constitutes one of the first applications of surfactant‐modified basal plane graphite electrodes to characterize the redox properties of a flavodoxin, thus providing a novel electrochemical method to study this class of protein. The X‐ray crystal structure of the flavodoxin purified from A. vinelandii was solved at 1.17 Å resolution. With this structure, the native nitrogenase electron transfer proteins have all been structurally characterized. Docking studies indicate that a common binding site surrounding the Fe‐protein [4Fe:4S] cluster mediates complex formation with the redox partners Mo‐Fe protein, ferredoxin I, and flavodoxin II. This model supports a mechanistic hypothesis that electron transfer reactions between the Fe‐protein and its redox partners are mutually exclusive.

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“…Experimentally, this hypothesis is supported by results from cross-linking studies and time-resolved limited proteolysis using NifF and Fe protein from A. vinelandii [16,52]. Similar modeling results were obtained for Fdx, although experimental support has not yet been reported [33,41].…”

“…It consists of a central five-stranded parallel β-sheet flanked on either side by α-helices. Based on the presence of a ∼20-residue loop splitting the fifth β-strand, Flds involved in electron transfer to nitrogenase are classified as long-chain flavodoxins [32,33]. The cofactor FMN has three pertinent redox states: oxidized quinone (Ox), semiquinone (SQ), and hydroquinone (HQ), where FMN Ox/SQ and FMN SQ/HQ couples are 1e − redox reactions.…”

“…[49,50] It is generally believed that in the Fe protein cycle, reduction of Fe protein and exchange of MgADPs for MgATPs takes place after its dissociation from MoFe protein, implying that the oxidized Fe protein cannot be reduced when bound to MoFe protein. Further, the Fe protein is believed to interact with Fld/Fdx using the same binding interface that is used with the MoFe protein [16,33,51,52]. This hypothesis is guided by docking models that predict electrostatic interactions between the positively charged surroundings of the [4Fe-4S] cluster of Fe protein and the negatively charged surroundings of FMN of NifF from A. vinelandii, possibly assisted by an eight amino acid loop (residues 64-71) on NifF [16,32,52] (Figure 2).…”

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“…mV/pH unit, respectively) with no redox-linked pK a observed in the range of 3.6 -7.3. This contrasts again with electrontransfer proteins, such as flavodoxins, that have a redox-linked pK a between pH 4.6 and 6.8 (25,27) (Fig. 5b).…”

“…Thus, the redox potential of FAD is not substantially changed by Fsq. This is in contrast to many enzymes and electron-transfer proteins that can alter the redox potentials more than 100 mV in either direction to carry out catalysis (25,26). There was relatively little difference in the effect of pH on the redox potential of free and complexed FAD (Ϫ45 mV/pH unit and Ϫ39…”