Primary sequence, oxidation‐reduction potentials and tertiary‐structure prediction of Desulfovibrio desulfuricans ATCC 27774 flavodoxin

Abstract

Flavodoxin was isolated and purified from Desulfovibrio desulfidricans ATCC 27774, a sulfatereducing organism that can also utilize nitrate as an alternative electron acceptor. Mid-point oxidation-reduction potentials of this flavodoxin were determined by ultravioletlvisible and EPR methods coupled to potentiometric measurements and their pH dependence studied in detail. The redox potential E2, for the couple oxidizedsemiquinone forms at pH 6.7 and 25°C is -40 mV, while the value for the semiquinonehydroquinone forms (El), at the same pH, -387 mV. E2 varies linearly with pH, while E, is independent of pH at high values. However, at low pH (<7.0), this value is less negative, compatible with a redox-linked protonation of the flavodoxin hydroquinone. Flavodoxins are a group of small electron-transfer proteins [l-31 (= 15-23 kDa) isolated from different organisms, that contain a single FMN group bound non-covalently to the polypeptide chain. They can transfer two electrons at low and differentiated redox potentials.The way in which a flavodoxin's peptide chain modulates the redox properties of the FMN cofactor has been the major focus of the tridimensional structural studies. Thermodynamic data have been discussed in terms of the binding of the cofactor to the polypeptide chain in different redox states of the protein. The pH dependence of the mid-point oxidationreduction potentials has been extensively explored.To infer on factors controlling the electron-transfer process, X-ray diffraction and NMR spectroscopy have been valuable tools to reveal. the environment of the FMN group bound to the protein. Anacystis nidulans [9] and Chondrus crispus [lo, 111 flavodoxins from X-ray studies ; and structures were proposed from two-dimensional and three-dimensional NMR studies for Megasphaera elsdenii [12-151 and D. vulgaris [16, 171 In this study we present the isolation, purification and characterization of D. desulfuricans ATCC 27774 flavodoxin. Redox potentials were determined by potentiometric titrations coupled with visible and EPR techniques, at different pH values. The value of the dissociation constant of the FMN group was estimated by differential spectrophotometric titrations of FMN with apo-flavodoxin. The complete amino acid sequence of D. desulfuricans ATCC 27774 flavodoxin was obtained and its degree of similarity with other Desulfovibrio species flavodoxins is presented. A theoretical model of a tertiary structure obtained using the most similar sequence available (D. vulgaris flavodoxin) is predicted using molecular-modeling tools. D. vulgaris

Effect of Inorganic Phosphate on FMN Binding and Loop Flexibility in Desulfovibrio desulfuricans Apo-flavodoxin

“…Also, FMN binding to Anabaena apo-flavodoxin differs from the D. vulgaris results: for this protein, FMN binding was shown to always start with isoalloxazine-ring interactions and the speed was independent of the presence or not of inorganic phosphate. 15 Here, we report on the FMN-binding mechanism in D. desulfuricans flavodoxin, which is highly homologous to the D. vulgaris species (44% sequence similarity 22 ). Despite the overall similarity in terms of sequence and fold, the K D for FMN binding is roughly 1000-fold higher to D. desulfuricans flavodoxin as compared to the D. vulgaris species.…”

1H and 15N NMR resonance assignments and solution secondary structure of oxidized Desulfovibrio desulfuricans flavodoxin

“…The secondary structure of D. desulfuricans flavodoxin, determined here by multinuclear NMR spectroscopy, is in good agreement with a model proposed by Caldeira et al (1994). The D. desulfuricans flavodoxin amino acid sequence reported by Caldeira et al (1994) is different, but highly homologous (79% identical), to that of the D. flavodoxin and the resulting structure was subjected to energy minimization.…”

“…The D. desulfuricans flavodoxin amino acid sequence reported by Caldeira et al (1994) is different, but highly homologous (79% identical), to that of the D. flavodoxin and the resulting structure was subjected to energy minimization. The NMR data presented here suggests that similar models can be built for other members of the Desulfovibrio family, which typically have about 50% sequence homology, with good reliability.…”

EPR and Mössbauer Spectroscopic Studies on Enoate Reductase

“…The intensity of the rhombic EPR signals does not increase upon further reduction with dithionite: the spin quantification (relative to a CuEDTA standard) gives 0.94 spin per monomer. The quantitation of the flavin signal is 0.9 spin per monomer, using as a standard Desulfovibrio desulfuricans ATCC 27774 flavodoxin in the semiquinone state at 120 K (20). The temperature dependence of the rhombic signal shows that it can be detected up to 60 K and is better observed at 10 K, using a microwave power of 23.7 W.…”