Crystal structure determination at 1.4 Å resolution of ferredoxin from the green alga Chlorella fusca

The low potential iron-sulfur (Fe-S) electron carriers known as ferredoxins (Fds) 1 are found in three distinct classes, the (7), and Aquifex aeolicus (8). The high level of similarity of these proteins allows for easy transfer of structural information among them. For instance, many properties of molecular variants of the C. pasteurianum [2Fe-2S] Fd (6, 9, 10) could be rationalized from the crystal structure of A. aeolicus Fd4 (11). This structure established the unexpected thioredoxin-like fold of these Fds and confirmed that they are distinct from the other two ferredoxin classes (1). The 2.3 Å resolution of the Fd4 structure, however, was not among the highest currently reported (about 1 Å) for metalloenzymes. Indeed, such high resolution structures are of utmost interest because they bring forth precise geometries of metal sites (4) and may allow description of redox transitions (5). Additional efforts have therefore been made, both on wild type (WT) thioredoxin-like Fds and on several of the molecular variants that were produced over the years (6,10,12), with the aim of improving the crystallographic data.In some modified forms of C. pasteurianum Fd, cysteine ligands of the Fe-S cluster were replaced by serine (9, 13). In their reduced ϩ level, these serine-ligated active sites were found to assume a delocalized mixed valence state having a ground spin state of 9/2 (14, 15). This unprecedented occurrence in binuclear iron-sulfur clusters has stimulated experimental and theoretical work (16,17). The development of these investigations has been hampered, however, by the absence of structural data on serine-ligated [2Fe-2S] active sites and their environment. Because only the Fd4 from A. aeolicus has been crystallized, we have repeated amino acid substitutions in that protein previously performed on C. pasteurianum Fd and produced the C55S and C59S variants that contain serine-ligated [2Fe-2S] clusters. We report here the high resolution structures for both of these variants as well as WT protein which provide accurate metric details for serine-ligated Fe-S clusters in proteins. EXPERIMENTAL PROCEDURESProtein Samples-Fd4 from A. aeolicus was purified as described by Chatelet et al. (8). The C55S and C59S variants were prepared by site-directed mutagenesis as described for the C56S and C60S counterparts from C. pasteurianum (13). The mutagenic oligonucleotides were

Section: Discussionmentioning

confidence: 99%

High Resolution Crystal Structures of the Wild Type and Cys-55 → Ser and Cys-59 → Ser Variants of the Thioredoxin-like [2Fe-2S] Ferredoxin from Aquifex aeolicus

Yeh

Ambroggio

Andrade

et al. 2002

“…Appropriated mediators that covered a potential range from ϩ11 mV to Ϫ450 mV (1 mM) (28) were added to the protein solution (10 M) in 50 mM Bis-Tris for pH 6.5 or 100 mM Tris-HCl for pH values 7.5 and 9. Injection of small volumes of air-free sodium dithionite allowed the reduction of the protein.…”

Section: Methodsmentioning

confidence: 99%

NADH Oxidase Activity of Mitochondrial Apoptosis-inducing Factor

Miramar

Costantini

Ravagnan

et al. 2001

349

278

Apoptosis-inducing factor (AIF) is a mitochondrial flavoprotein, which translocates to the nucleus during apoptosis and causes chromatin condensation and large scale DNA fragmentation. Here we report the biochemical characterization of AIF's redox activity. Natural AIF purified from mitochondria and recombinant AIF purified from bacteria (AIF⌬1-120) exhibit NADH oxidase activity, whereas superoxide anion (O 2 ؊ ) is formed. AIF⌬1-120 is a monomer of 57 kDa containing 1 mol of noncovalently bound FAD/mol of protein. ApoAIF⌬1-120, which lacks FAD, has no NADH oxidase activity. However, native AIF⌬1-120, apoAIF⌬1-120, and the reconstituted (FAD-containing) holoAIF⌬1-120 protein exhibit a similar apoptosis-inducing potential when microinjected into the cytoplasm of intact cells. Inhibition of the redox function, by external addition of superoxide dismutase or covalent derivatization of FAD with diphenyleneiodonium, failed to affect the apoptogenic function of AIF⌬1-120 assessed on purified nuclei in a cellfree system. Conversely, blockade of the apoptogenic function of AIF⌬1-120 with the thiol reagent parachloromercuriphenylsulfonic acid did not affect its NADH oxidase activity. Altogether, these data indicate that AIF has a marked oxidoreductase activity which can be dissociated from its apoptosis-inducing function.

“…(38) using a function that fitted the amino acid sequence of FNR to that of the CooA monomer followed by energy minimization and further fitting to minimize the root mean square (between 1 and 2 Å) (16, 34, 36). The 2Fe cluster was fitted using the [2Fe-2S] cluster of the oxidized form of ferredoxin from Chlorella fusca (1AWD.pdb) (39).…”

Section: Product Of Oxygen Reduction By Fnr-mentioning

confidence: 99%

Mechanism of Oxygen Sensing by the Bacterial Transcription Factor Fumarate-Nitrate Reduction (FNR)

Crack¹,

Green

Thomson³

2004

125

109

The facultative anaerobe Escherichia coli adopts different metabolic modes in response to the availability of oxygen. The global transcriptional regulator FNR (fumarate-nitrate reduction) monitors the availability of oxygen in the environment. Binding as a homodimer to palindromic sequences of DNA, FNR carries a sensory domain, remote from the DNA binding helix-turn-helix motif, which responds to oxygen. 2؉ , causing a similar cluster transformation to a [2Fe-2S] form. Either the hydrogen peroxide formed on reaction with oxygen can be recycled by intracellular catalase or it can be used to oxidize further Fe-S clusters. In both cases, the efficacy of oxygen sensing by FNR will be increased.Escherichia coli is a facultative anaerobe that adopts different metabolic modes in response to the availability of oxygen (1). A hierarchy of metabolism exists in which aerobic respiration is preferred to anaerobic respiration, which in turn is preferred to fermentation (1). In simple terms, the global transcriptional regulator FNR 1 (designated due to defects in fumarate-nitrate reduction of the corresponding mutants), along with other transcription factors, ArcBA, NarXL, NarPQ, and FhlA, maintains this hierarchy by monitoring the availability of oxygen in the environment (1). FNR is a member of a large family of transcription factors that modulate physiological changes in response to a variety of metabolic and environmental challenges (2). Members of the family are predicted to be structurally related to the catabolite gene activator protein, CAP (also known as the cAMP receptor protein). CAP and FNR proteins bind as homodimers to palindromic sequences of DNA, each monomer binding to one half-site (2). They consist of two functionally distinct domains, a DNA binding helix-turn-helix motif and an N-terminal region of antiparallel ␤-strands forming the sensory domain (3). The sensing regions are adapted to respond to different effectors (2). Thus, CAP reversibly binds cAMP to monitor glucose status (4), and the sensing domain of the CooA protein of Rhodospirillum rubrum possesses a b-type cytochrome that binds CO (5). The sensory region of FNR has four conserved cysteine residues that are essential for in vivo activity and capable of ligating an oxygen-sensitive [4Fe-4S] iron-sulfur cluster (6 -11). A variety of studies have revealed that the active form of FNR contains one [4Fe-4S] 2ϩ cluster/protein monomer that is converted to a [2Fe-2S] 2ϩ cluster, together with other, less well defined iron species, following exposure to oxygen both in vitro and in vivo (10,(12)(13)(14)(15). The switch from a cubane [4Fe-4S] cluster, bound to the protein by four cysteine thiol ligands that sit at the vertices of a tetrahedron, to a planar [2Fe-2S] cluster, also thought to possess four cysteine ligands but lying in a plane, suggests that cluster transformation on exposure to oxygen will provide a large conformational change in the N-terminal region of FNR, presumably thereby initiating the switch of the protein from a DNA binding state...