Lars Skjeldal

Structure-function studies of [2Fe-2S] ferredoxins

The ability to overexpress [2Fe-2S] ferredoxins inEscherichia coli has opened up exciting research opportunities. High-resolution x-ray structures have been determined for the wild-type ferredoxins produced by the vegetative and heterocyst forms ofAnabaena strain 7120 (in their oxidized states), and these have been compared to structural information derived from multidimensional, multinuclear NMR spectroscopy. The electron delocalization in these proteins in their oxidized and reduced states has been studied by1H,2H,13C, and15N NMR spectroscopy. Site-directed mutagenesis has been used to prepare variants of these ferredoxins. Mutants (over 50) of the vegetative ferredoxin have been designed to explore questions about cluster assembly and stabilization and to determine which residues are important for recognition and electron transfer to the redox partnerAnabaena ferredoxin reductase. The results have shown that serine can replace cysteine at each of the four cluster attachment sites and still support cluster assembly. Electron transfer has been demonstrated with three of the four mutants. Although these mutants are less stable than the wild-type ferredoxin, it has been possible to determine the x-ray structure of one (C49S) and to characterize all four by EPR and NMR. Mutagenesis has identified residues 65 and 94 of the vegetative ferredoxin as crucial to interaction with the reductase. Three-dimensional models have been obtained by x-ray diffraction analysis for several additional mutants: T48S, A50V, E94K (four orders of magnitude less active than wild type in functional assays), and A43S/A45S/T48S/A50N (quadruple mutant).

A putative DNA-binding domain in the NUCKS protein

We have studied the DNA-binding properties of a NUCKS-derived, synthetic peptide containing an extended GRP motif. This peptide binds to random-sequence DNA, but did not bind preferentially to poly(dA-dT). A synthetic peptide with the same amino acid composition but with a random sequence did not bind to DNA, suggesting that the structure of the DNA-binding domain plays a pivotal role in the interaction with DNA. NMR and graphic modeling were employed to investigate the structure of the synthetic peptide. It was shown that the DNA-binding peptide constituted an alpha helix in phosphate buffer at pH 5.5. Docking results indicated an almost perfect fit for this small, helical peptide into the major groove of DNA with the possibility of four basic residues interacting with the phosphate backbone of DNA. One consensus site for phosphorylation by Cdk1 is located in the N-terminal end of the DNA-binding peptide. Upon phosphorylation of this site, the binding to DNA was completely prohibited. Immunofluorescence experiments showed that NUCKS was located in the nuclei in proliferating cells in interphase of the cell cycle, but was distributed throughout the cytoplasm in mitotic cells.

Carbon monoxide dehydrogenase from Rhodospirillum rubrum produces formate

Abstract. Carbon monoxide dehydrogenase (CODH) from Rhodospirillumrubrum reversibly catalyzes the oxidation of CO to CO2 at the active site C-cluster. In this article, the reduction of CO2 to formate is reported as a slow side reaction catalyzed by both Ni-containing CODH and Ni-deficient CODH. Recently, the structures of R. rubrum CODH and its active site NiFeS cluster (the C-cluster) have been solved. The data in this manuscript describe the formate-producing capability of CODH with or without Ni in the active site.

Detection and characterization of hyperfine-shifted resonances in the proton nuclear magnetic resonance spectrum of Anabaena 7120 ferredoxin at high magnetic fields

This paper presents previously unobserved signals in the 1H NMR spectra of oxidized and reduced [2Fe-2S]-ferredoxin from Anabaena 7120 detected at 400, 500, and 600 MHz. The signals shifted to low field exhibited longitudinal relaxation (T1) values in the range of 100-400 microseconds and line widths in the range of 1-10 kHz (at 400 MHz), and the chemical shifts of all signals showed strong temperature dependence. Although the line widths were smaller at lower magnetic fields, the resolution was better at higher magnetic fields. In the oxidized state, a broad signal was detected at 37 ppm, which corresponds to at least 6 protons, and whose chemical shift exhibits positive temperature dependence. This signal also was found in oxidized ferredoxin reconstituted in 2H2O, which excludes the signal as arising from solvent-exchangeable amide protons. In the reduced state, four signals detected between 90 and 140 ppm exhibited negative temperature dependence. These consisted of two pairs of signals, each pair having one component with half the linewidth of the other. On the basis of their chemical shifts, linewidths, longitudinal relaxation properties, and temperature dependence we assigned these resonances to four of the beta hydrogens of the ligated cysteines. Two solvent-exchangeable hyperfine-shifted signals were found in the reduced state; these are located upfield of the diamagnetic region. The low-field hyperfine resonances of half-reduced ferredoxin in the presence of sodium dithionite showed a self electron transfer exchange rate that was slow on the NMR scale as observed earlier (Chan, T., and Markley, J. L. (1983) Biochemistry 22, 5982-5987), but the exchange rate was accelerated in the presence of methyl viologen.

Letter to the Editor: Assignment of 1H, 13C and 15N NMR signals from toluene 4-monooxygenase Rieske ferredoxin in its oxidized state

Rieske [2Fe-2S] centers are found in membrane ubiquinone cytochrome oxidase complexes (Trumpower and Gennis, 1994), as integral parts of the active site in the cis-dihydrodiol forming aromatic dioxygenases (Mason and Cammack, 1992), and as soluble electron carriers in bacterial dioxygenase and monooxygenase complexes (Harayama et al., 1992). X-ray crystal structures of the Rieske domains from the bovine bc1 (Iwata et al., 1996; Link and Iwata, 1996) and chloroplastic b6f oxidase (Carrell et al., 1997) complexes, naphthalene dioxygenase (Kauppi et al., 1998), and the soluble electron carrier ferredoxin of the Burkholderia sp. strain LB400 biphenyl dioxygenase (Colbert et al., 2000) have been reported. A more comprehensive understanding of the functional specialization of the Rieske-type ferredoxins would be advanced by the availability of additional structural and functional information. Toluene 4-monooxygenase (T4MO) from Pseudomonas mendocina is a soluble bacterial monooxygenase complex (Fox, 1998), consisting of an NADH oxidoreductase (T4moF), a diiron hydroxylase [T4moH, (αβγ)2 quaternary structure (Pikus et al., 1996)], a catalytic effector protein [T4moD (Hemmi et al., 2001)], and a Rieske ferredoxin (T4moC, 12 195 Da after removal of N-terminal Met). T4moC acts as an obligate electron carrier between T4moF and T4moH. Here we report the assignment and deposition of diamagnetic chemical shifts for oxidized T4moC. The solution structure of T4moC arising from these

Multinuclear Magnetic Resonance and Mutagenesis Studies of Structure-Function Relationships in [2Fe-2S] Ferredoxins

Iron-sulfur proteins are present in virtually all living organisms. Their function is to transfer electrons to various partners. They have iron and sulfur in their chromophore and rather low molecular weights (5 to 25 kDa). They usually have the same ratio of iron to sulfur except for rubredoxin which has only one iron ligated by four cysteine residues. Several cluster classes have been identified in iron-sulfur proteins, including [1Fe], [2Fe-2S], [4Fe-4S], and [3Fe-4S] types (Figure 1). Some iron-sulfur proteins contain more than one cluster of a given type or of different types. [2Fe-2S] ferredoxins contain two high-spin ferric ions that are antiferromagnetically coupled resulting a total spin number of zero in the oxidized state; thus they are EPR1 silent. Population of higher electronic states, however, makes the cluster paramagnetic at room temperature (and at all temperature studied by NMR). Upon reduction, one of the two ferric ions (Fe3+) is changed to a ferrous ion (Fe2+), and the other remains ferric. Reduced [2Fe-2S] ferredoxins have a total spin number of one-half and produce an EPR signal. [4Fe-4S] cluster types have three possible oxidation states: 3Fe3+-Fe2+, 2Fe3+-2Fe2+, and Fe3+-3Fe2+. Typically in iron-sulfur proteins, only two of the three states are easily accessible: the first two as in high-potential iron-sulfur proteins (HiPIPs) or the last two as in [4Fe-4S] ferredoxins. We have recently investigated three different [2Fe-2S] ferredoxins by NMR spectroscopy: Anabaena 7120 vegetative ferredoxin, Anabacna 7120 heterocyst ferredoxin, and human placental ferredoxin. The first two are plant-type ferredoxins from a cyanobacterium, and the last is vertebrate-type ferredoxin. Open image in new window Figure 1. Four different cluster types appearing in iron-sulfur proteins. The first three involve ligation of the cluster to four cysteinc residues of the protein chain. One cysteine is absent in the fourth cluster type. (A) One Fe ligated by four cysteines; (B) [2Fe-2S] cluster; (C) [4Fe-4S] cluster; (D) [3Fe-4S] cluster.