A J Thomson

Does ferredoxin I (Azotobacter) represent a novel class of DNA-binding proteins that regulate gene expression in response to cellular iron(II)?

Azotobacter vinelandii (Av) and chroococcum (Ac) ferredoxin I contain [3F‐4S]1+/0 and [4Fe‐4S]2+/1+ clusters, when isolated aerobically, which undergo one‐electron redox cycles at potentials of −460±10 mV (vs SHE) at pH 8.3 and −645± 10 mV, respectively. The X‐ray structure of Fd I (Av) reveals that the N‐terminal half of the polypeptide folds as a sandwich of β‐strands which enclose the iron‐sulphur clusters. The C‐terminal sequence contains an amphiphilic α‐helix of four turns which lies on the surface of the β‐barrel. Fd I (Av) controls expression of an unknown protein of Mr ∼ 18 000. Fd I (Ac) will complex iron(II) avidly above pH ∼ 8.0 only when the [3Fe‐4S] cluster is reduced and provided that cellular nucleic acid is bound. Fd I (Ac) rigorously purified from nucleic acid does not undergo iron(II) uptake. These facts, together with recent evidence that the interconversion process [3Fe‐4S]0 + Fe2+ → [4Fe‐4S]2+ in the iron‐responsive element binding protein (IRE‐BP) of eukaryotic cells is controlling protein expression at the level of mRNA (1991, Cell 64, 4771; 1991, Nucleic Acid Res. 19, 1739] leads to the following hypothesis. Fd I is a DNA‐binding protein which interacts by single α‐helix binding in the wide groove of DNA. The binding is regulated by iron(II) levels in the cell. The 7Fe form binds to DNA and represses gene expression. Only the DNA‐bound form of the 7Fe Fd I will take up iron(II), not the form free in solution. Iron(II) becomes bound when the [3Fe‐4S] cluster is reduced. The 8Fe Fd I thus generated no longer binds DNA and the gene is de‐repressed. Sequence comparisons and the crystal structure suggests that the two central turns of the α‐helix are important elements of the DNA‐recognition process and that residues Gln69 and Glu73, which lie on the outer surface of the helix, hydrogen‐bond with specific base pairs.

Thiocyanate binding to the molybdenum centre of the periplasmic nitrate reductase from Paracoccus pantotrophus.

The periplasmic nitrate reductase (NAP) from Paracoccus pantotrophus is a soluble two-subunit enzyme (NapAB) that binds two haem groups, a [4Fe-4S] cluster and a bis(molybdopterin guanine dinucleotide) (MGD) cofactor that catalyses the reduction of nitrate to nitrite. In the present study the effect of KSCN (potassium thiocyanate) as an inhibitor and Mo ligand has been investigated. Results are presented that show NAP is sensitive to SCN(-) (thiocyanate) inhibition, with SCN(-) acting as a competitive inhibitor of nitrate (K(i) approximately 4.0 mM). The formation of a novel EPR Mo(V) species with an elevated g(av) value (g(av) approximately 1.994) compared to the Mo(V) High-g (resting) species was observed upon redox cycling in the presence of SCN(-). Mo K-edge EXAFS analysis of the dithionite-reduced NAP was best fitted as a mono-oxo Mo(IV) species with three Mo-S ligands at 2.35 A (1 A=0.1 nm) and a Mo-O ligand at 2.14 A. The addition of SCN(-) to the reduced Mo(IV) NAP generated a sample that was best fitted as a mono-oxo (1.70 A) Mo(IV) species with four Mo-S ligands at 2.34 A. Taken together, the competitive nature of SCN(-) inhibition of periplasmic nitrate reductase activity, the elevated Mo(V) EPR g(av) value following redox cycling in the presence of SCN(-) and the increase in sulphur co-ordination of Mo(IV) upon SCN(-) binding, provide strong evidence for the direct binding of SCN(-) via a sulphur atom to Mo.

The design and sensitivity of microwave frequency optical heterodyne receivers

Recent advances in high speed photodetector and microwave receiver technology make microwave frequency optical heterodyning an attractive approach for the detection of a number of coherent Raman and Brillouin scattering experiments. We have therefore analyzed the sensitivity of microwave frequency optical heterodyne receivers. Experimental tests on a visible wavelength receiver operating at 13.5 GHz confirm the expectation of shot noise limited sensitivity. The relative merits of microwave frequency optical heterodyne detection and the alternative Fabry–Perot interferometry approach are discussed.

Spectroscopic studies of partially reduced forms of Wolinella succinogenes nitrite reductase.

Reductive titrations of the dissimilatory hexa‐haem nitrite reductase, Wolinella succinogenes, with methyl viologen semiquinone (MV) and sodium dithionite, have been followed at room temperature by absorption, natural (CD) and magnetic circular dichroism (MCD) spectroscopies and at liquid helium temperature by electron paramagnetic resonance (EPR) and MCD spectroscopies. The nature of the reduced enzyme depends on the reductant employed. At room temperature a single high‐spin ferous haem, observed by MCD after reduction with MV, is absent from dithionite reduced samples. It is suggested that a product of dithionite oxidation becomes bound with high affinity to the reduced state of the enzyme causing the ferrous haem to become low‐spin. The site occupied is likely to be the substrate binding haem. The course of the titration with MV at room temperature shows the reduction of high‐spin ferric to high‐spin ferrous haem. Since the EPR spectrum reveals the presence of an unusual high‐low spin ferric haem pair in the oxidised state we propose that the active site of the enzyme is a novel haem pair consisting of one high (5‐coordinate) and one low‐spin (6 coordinate) haem, magnetically coupled and possibly bridged by a histidinate ligand.

Characterization of the oxidized P-clusters in the MoFe protein of Klebsiella pneumoniae nitrogenase by MCD spectroscopy.

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