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Abstract

Recombinant Desulfovibrio vulgaris flavodoxin was produced in Escherichia coli. A complete backbone NMR assignment for the two-electron reduced protein revealed significant changes of chemical shift values compared to the oxidized protein, in particular for the flavine mononucleotide (FMN)-binding site. A comparison of homo- and heteronuclear NOESY spectra for the two redox states led to the assumption that reduction is not accompanied by significant changes of the global fold of the protein. The backbone dynamics of both the oxidized and reduced forms of D. vulgaris flavodoxin were investigated using two-dimensional 15N-1H correlation NMR spectroscopy. T1, T2 and NOE data are obtained for 95% of the backbone amide groups in both redox states. These values were analysed in terms of the 'model-free' approach introduced by Lipari and Szabo [(1982) J. Am. Chem. Soc., 104, 4546-4559, 4559-4570]. A comparison of the two redox states indicates that in the reduced species significantly more flexibility occurs in the two loop regions enclosing FMN. Also, a higher amplitude of local motion could be found for the N(3)H group of FMN bound to the reduced protein compared to the oxidized state.

Conformational Dynamics of Escherichia coli Flavodoxins in Apo- and Holo-States by Solution NMR Spectroscopy

“…Further experimental data suggested that lacking of the flexible glycine residue in the 50 s loop would severely affect the redox potential, thus supporting the importance of the conformational flexibility of this loop in the redox reactions [56]. Our current backbone dynamics studies of holo-YqcA and holo-FldA, together with previous reports on MioC [17] and D. Vulgaris flavodoxin [51], demonstrate that the µs-ms timescale conformational exchanges in the 50 s loop are commonly observed in the holo-form of both long-chain and short-chain flavodoxins. Since the holo-proteins used in these studies all contain oxidized FMN molecule, the observed conformational dynamics suggest that the 50 s loop samples multiple conformational spaces in this single redox state, and the local conformational flexibility facilitates the fine-tuning and adaption of the protein structure to other redox states during electron transfer.…”

“…Since flavodoxins are a model system for studies of protein folding and cofactor binding, they have been subjected to various biochemical and structural characterizations, including several early dynamics investigations by solution NMR technique [49]–[51]. The backbone dynamics study of the Desulfovibrio vulgaris flavodoxin by Hrovat et al also revealed higher than average R 2 /R 1 values at the 50 s loop region, which is also an indication of conformational exchanges on the slow timescales [51]. Intriguingly, mutagenesis and crystallographic studies showed that the flexibility of the 50 s loop plays an important role in the redox redactions of flavodoxins.…”

Mapping Solvation Dynamics at the Function Site of Flavodoxin in Three Redox States

“…From the x-ray structure,19 our MD simulations (Fig. 5C), and the NMR studies,45 the shallow binding pocket become looser, mainly in the regions of 56–82 residues of the 60’s loop and 91–103 residues of the 90’s loop and largely due to the repulsion between FMNH – and the surrounding negatively-charged residues such as D62 and D95 (Fig. 1 and Fig.…”

Distant residues mediate picomolar binding affinity of a protein cofactor

“…As a result, flavodoxin can transfer electrons between various physiological redox partners. Strong binding of FMN is essential for flavodoxins to be functionally active, and these proteins are remarkably rigid on the ns-timescale and below19202122. The isoalloxazine moiety and 5′-phosphate group of FMN contribute almost equally to the free energy associated with cofactor binding, whereas the ribityl has barely any impact23.…”