I. Lane

X-ray absorption spectroscopy of dimethylsulfoxide reductase from Rhodobacter capsulatus

Abstract Mo K-edge X-ray absorption spectroscopy (XAS) has been used to probe the environment of Mo in dimethylsulfoxide (DMSO) reductase from Rhodobacter capsulatus in concert with protein crystallographic studies. The oxidised (MoVI) protein has been investigated in solution at 77 K; the Mo K-edge position (20006.4 eV) is consistent with the presence of MoVI and, in agreement with the protein crystallographic results, the extended X-ray absorption fine structure (EXAFS) is also consistent with a seven-coordinate site. The site is composed of one oxo-group (Mo=O 1.71 Å), four S atoms (considered to arise from the dithiolene groups of the two molybdopterins, two at 2.32 Å and two at 2.47 Å, and two O atoms, one at 1.92 Å (considered to be H-bonded to Trp 116) and one at 2.27 Å (considered to arise from Ser 147). The Mo K-edge XAS recorded for single crystals of oxidised (MoVI) DMSO reductase at 77 K showed a close correspondence to the data for the frozen solution but had an inferior signal:noise ratio. The dithionite-reduced form of the enzyme and a unique form of the enzyme produced by the addition of dimethylsulfide (DMS) to the oxidised (MoVI) enzyme have essentially identical energies for the Mo K-edge, at 20004.4 eV and 20004.5 eV, respectively; these values, together with the lack of a significant presence of MoV in the samples as monitored by EPR spectroscopy, are taken to indicate the presence of MoIV. For the dithionite-reduced sample, the Mo K-edge EXAFS indicates a coordination environment for Mo of two O atoms, one at 2.05 Å and one at 2.51 Å, and four S atoms at 2.36 Å. The coordination environment of the Mo in the DMS-reduced form of the enzyme involves three O atoms, one at 1.69 Å, one at 1.91 Å and one at 2.11 Å, plus four S atoms, two at 2.28 Å and two at 2.37 Å. The EXAFS and the protein crystallographic results for the DMS-reduced form of the enzyme are consistent with the formation of the substrate, DMSO, bound to MoIV with an Mo-O bond of length 1.92 Å.

Characterization of a molybdenum cofactor biosynthetic gene cluster in Rhodobacter capsulatus which is specific for the biogenesis of dimethylsulfoxide reductase

The DMSO reductase of Rhodobacter capsulatus contains a pterin molybdenum cofactor (Moco) and is located in the periplasm. DNA sequence analysis identified four genes involved in the biosynthesis of the Moco (moaA, moaD, moeB and moaC) immediately downstream of the dor (DMSO respiratory) gene cluster. Rhodobacter capsulatus MoaA was expressed in Escherichia coli as a His6-tagged protein. Although, the expressed protein formed inclusion bodies, EPR spectroscopy showed that MoaA contains a [3Fe-4S] cluster. A moaA mutant was constructed and its phenotype indicates that the Moco biosynthetic gene cluster downstream of the dor operon is specific for the biogenesis of DMSO reductase. Two forms of DMSO reductase were purified by immunoaffinity chromatography from the moaA mutant. A mature form of DMSO reductase was located in the periplasm and a precursor form was found in the cytoplasm.

Tolyporphin Macrocycles from the Cyanobacterium Tolypothrix nodosa Selectively Bind Copper and Silver and Reverse Multidrug Resistance

Tolyporphins are glycosylated macrocycles isolated from lipophilic soil extracts of the cyanobacterium, Tolypothrix nodosa, and found to potentiate the cytotoxicity of antitumor drugs like vinblastine and adriamycin. Here we find that, unlike porphyrins, tolyporphins are not able to form complexes with most metal ions. However, they do react strongly with copper(II) and silver(II), forming square-planar metal complexes with an unpaired electron in a dx2-y2 orbital of the metal delocalized onto the ligating tolyporphin nitrogen atoms. Complexes were characterized by visible absorption spectra, mass spectrometry (EI, FAB, ESI, LDI-TOF, and MALDI-TOF) and multifrequency continuous-wave electron paramagnetic resonance spectra. Copper(II) and silver(II) complexes of tolyporphins A and E were found to have the interesting property of reversing multidrug resistance (MDR), with the copper complexes being less toxic than free tolyporphins. Reactive oxygen-free radicals were implicated in both the cytotoxic and MDR-reversing effects of free and metalated tolyporphins.

Dimethylsulfoxide (DMSO) reductase a member of the DMSO reductase family of molybdenum enzymes

Herein we describe the application of continuous wave (CW) and pulsed EPR spectroscopy to the structural (geometric and electronic) characterization of the Mo(V) active site within dimethylsulfoxide (DMSO) reductase and where appropriate, model molybdenum(V) complexes. Specifically, the electronic and geometric structure of the Low-g and High-g EPR signals and their relevance to the enzymes’ catalytic cycle are described. CW and pulsed EPR studies of a dithionite-reduced sample of DMSO reductase reveal the presence of a sulfur-centered radical localized on the molybdenum cofactor.

Structural characterisation of the Mo(V) High-g unsplit species from Rhodobacter capsulatus dimethylsulfoxide reductase

Dimethyl sulfoxide reductase of Rhodobacter capsulatus contains a bis(molybdopterin guanine dinucleotide molybdenum cofactor (bis-MGD-Mo) and can reduce dimethylsulfoxide to dimethylsulfide and trimethylamine-N-oxide to trimethylamine. Tryptophan-I 16 forms a hydrogen bond with the single 0x0 ligand coordinated to the molybdenum ion. Optical and EPR redox potentiometric titrations identified a single MO(V) species, the high-g unsplit species with Mo(VUV) and Mo(V/IV) redox potentials of +155, +60 mV at pH 8.0 with respect to the standard hydrogen elecrode. Multifrequency EPR studies ofthe 95Mo enriched enzyme allowed the accurate determination of the g5Mo hyperfme matrix which in conjunction with the g matrix from computer simulation of the EPR spectra of the native enzyme, shows that the unpaired electron is in a predominantly dZ based molecular orbital. Orientation selective HYSCORE measurements reveal the presence of proton and nitrogen hyperfine coupling arising from coupling of the unpaired electron with the proton and nitrogen on tryptophan-116.