Wilfred R. Hagen

Characterization of the Corrinoid Iron-Sulfur Protein Tetrachloroethene Reductive Dehalogenase of Dehalobacter restrictus

ABSTRACT The membrane-bound tetrachloroethene reductive dehalogenase (PCE-RDase) (PceA; EC 1.97.1.8), the terminal component of the respiratory chain of Dehalobacter restrictus, was purified 25-fold to apparent electrophoretic homogeneity. Sodium dodecyl sulfate-polyacrylamide gel electrophoresis revealed a single band with an apparent molecular mass of 60 ± 1 kDa, whereas the native molecular mass was 71± 8 kDa according to size exclusion chromatography in the presence of the detergent octyl-β-d-glucopyranoside. The monomeric enzyme contained (per mol of the 60-kDa subunit) 1.0± 0.1 mol of cobalamin, 0.6 ± 0.02 mol of cobalt, 7.1± 0.6 mol of iron, and 5.8 ± 0.5 mol of acid-labile sulfur. Purified PceA catalyzed the reductive dechlorination of tetrachloroethene and trichloroethene to cis-1,2-dichloroethene with a specific activity of 250 ± 12 nkat/mg of protein. In addition, several chloroethanes and tetrachloromethane caused methyl viologen oxidation in the presence of PceA. The Km values for tetrachloroethene, trichloroethene, and methyl viologen were 20.4± 3.2, 23.7 ± 5.2, and 47 ± 10 μM, respectively. The PceA exhibited the highest activity at pH 8.1 and was oxygen sensitive, with a half-life of activity of 280 min upon exposure to air. Based on the almost identical N-terminal amino acid sequences of PceA of Dehalobacter restrictus, Desulfitobacterium hafniense strain TCE1 (formerly Desulfitobacterium frappieri strain TCE1), and Desulfitobacterium hafniense strain PCE-S (formerly Desulfitobacterium frappieri strain PCE-S), the pceA genes of the first two organisms were cloned and sequenced. Together with the pceA genes of Desulfitobacterium hafniense strains PCE-S and Y51, the pceA genes of Desulfitobacterium hafniense strain TCE1 and Dehalobacter restrictus form a coherent group of reductive dehalogenases with almost 100% sequence identity. Also, the pceB genes, which may code for a membrane anchor protein of PceA, and the intergenic regions of Dehalobacter restrictus and the three desulfitobacteria had identical sequences. Whereas the cprB (chlorophenol reductive dehalogenase) genes of chlorophenol-dehalorespiring bacteria are always located upstream of cprA, all pceB genes known so far are located downstream of pceA. The possible consequences of this feature for the annotation of putative reductive dehalogenase genes are discussed, as are the sequence around the iron-sulfur cluster binding motifs and the type of iron-sulfur clusters of the reductive dehalogenases of Dehalobacter restrictus and Desulfitobacterium dehalogenans identified by electron paramagnetic resonance spectroscopy.

Redox chemistry of cobalamin and iron-sulfur cofactors in the tetrachloroethene reductase of Dehalobacter restrictus

Respiration of Dehalobacter restrictus is based on reductive dechlorination of tetrachloroethene. The terminal component of the respiratory chain is the membrane‐bound tetrachloroethene reductase. The metal prosthetic groups of the purified enzyme have been studied by optical and EPR spectroscopy. The 60‐kDa monomer contains one cobalamin with E m(Co1+/2+)=−350 mV and E m(Co2+/3+)>150 mV and two electron‐transferring [4Fe–4S](2+;1+) clusters with rather low redox potentials of E m≈−480 mV. The cob(II)alamin is present in the base‐off configuration. A completely reduced enzyme sample reacted very rapidly with tetrachloroethene yielding base‐off cob(II)alamin rather than trichlorovinyl‐cob(III)alamin.

The catalytic center of ferritin regulates iron storage via Fe(II)-Fe(III) displacement.

A conserved iron-binding site, the ferroxidase center, regulates the vital iron storage role of the ubiquitous protein ferritin in iron metabolism. It is commonly thought that two Fe(II) simultaneously bind the ferroxidase center and that the oxidized Fe(III)-O(H)-Fe(III) product spontaneously enters the cavity of ferritin as a unit. In contrast, in some bacterioferritins and in archaeal ferritins a persistent di-iron prosthetic group in this center is believed to mediate catalysis of core formation. Using a combination of binding experiments and isotopically labeled (57)Fe(II), we studied two systems in comparison: the ferritin from the hyperthermophilic archaeal anaerobe Pyrococcus furiosus (PfFtn) and the eukaryotic human H ferritin (HuHF). The results do not support either of the two paradigmatic models; instead they suggest a unifying mechanism in which the Fe(III)-O-Fe(III) unit resides in the ferroxidase center until it is sequentially displaced by Fe(II).

A novel S=3/2 EPR signal associated with native Fe-proteins of nitrogenase

In addition to their g = 1.94 EPR signal, nitrogenase Fe‐proteins from Azotobacter vinelandii, Azotobacter chroococcum and Klebsiella pneumoniae exhibit a weak EPR signal with g ≅5. Temperature dependence of the signal was consistent with an S = system with negative zero‐field splitting, d = −5 ± 0.7 cm−1. The m s, = ± ground state doublet gives rise to a transition with g eff = 5.90 and the transition within the excited m s = ± doublet has a split g eff = 4.8, 3.4. Quantitation gave 0.6 to 0.8 spin mol−1 which summed with the spin intensity of the S = g = 1.94 line to roughly 1 . MgATP and MgADP decreased the intensity of the s = signal with no concomitant changes in intensity of the s = signal.

Oleate Hydratase Catalyzes the Hydration of a Nonactivated Carbon-Carbon Bond

The hydration of oleic acid into 10-hydroxystearic acid was originally described for a Pseudomonas cell extract almost half a century ago. In the intervening years, the enzyme has never been characterized in any detail. We report here the isolation and characterization of oleate hydratase (EC 4.2.1.53) from Elizabethkingia meningoseptica.

Tungsten Transport Protein A (WtpA) in Pyrococcus furiosus: the First Member of a New Class of Tungstate and Molybdate Transporters

A novel tungstate and molybdate binding protein has been discovered from the hyperthermophilic archaeon Pyrococcus furiosus. This tungstate transport protein A (WtpA) is part of a new ABC transporter system selective for tungstate and molybdate. WtpA has very low sequence similarity with the earlier-characterized transport proteins ModA for molybdate and TupA for tungstate. Its structural gene is present in the genome of numerous archaea and some bacteria. The identification of this new tungstate and molybdate binding protein clarifies the mechanism of tungstate and molybdate transport in organisms that lack the known uptake systems associated with the ModA and TupA proteins, like many archaea. The periplasmic protein of this ABC transporter, WtpA (PF0080), was cloned and expressed in Escherichia coli. Using isothermal titration calorimetry, WtpA was observed to bind tungstate (dissociation constant [K(D)] of 17 +/- 7 pM) and molybdate (K(D) of 11 +/- 5 nM) with a stoichiometry of 1.0 mol oxoanion per mole of protein. These low K(D) values indicate that WtpA has a higher affinity for tungstate than do ModA and TupA and an affinity for molybdate similar to that of ModA. A displacement titration of molybdate-saturated WtpA with tungstate showed that the tungstate effectively replaced the molybdate in the binding site of the protein.

The iron-sulfur composition of the active site of hydrogenase from Desulfovibrio vulgaris (Hildenborough) deduced from its subunit structure and total iron-sulfur content

Hydrogenase Iron‐sulfur cluster (Desulfovibrio vulgaris)

Novel structure and redox chemistry of the prosthetic groups of the iron-sulfur flavoprotein sulfide dehydrogenase from Pyrococcus furiosus; evidence for a [2Fe-2S] cluster with Asp(Cys) (3) ligands

2]2 − and its ability to bind to biological macromolecules should not be overlooked, and may artificially trigger/accelerate Cu(II) reduction.

Quantitative Numerical Analysis of g Strain in the EPR of Distributed Systems and Its Importance for Multicenter Metalloproteins

A method for simulation of inhomogeneously broadened EPR of metallo-proteins based on recent theoretical advances is surveyed critically in terms of efficiency and accuracy. From the quality of the experimental spectrum, minimal boundary conditions are established for the spatial integration over the g-strained polycrystal. Computational efficiency is achieved by generating the S = 12 spectrum as an absorption in g space, reducing the number of molecular orientations computed by filtering mosaic artifacts from the Fourier-transformed spectrum, and generating the lineshape due to g strain from a tabulated distribution function. These techniques provide a reduction in computation time by some two orders of magnitude and make the data analysis of EPR of metalloproteins by minimization practical. The resulting simulation program is superior to current approaches in that it does not introduce artifactual multiplicities, and it is expected to require a smaller number of fitting parameters for the quantitative analysis of most cases. To illustrate its potential, the method is applied to EPR data from the iron-sulfur centers in NADH:Q oxidoreductase and in QH2:ferricytochrome c oxidoreductase, clarifying existing controversies on the stoichiometries of these centers.

Characterization of the Chlorate Reductase from Pseudomonas chloritidismutans

A chlorate reductase has been purified from the chlorate-reducing strain Pseudomonas chloritidismutans. Comparison with the periplasmic (per)chlorate reductase of strain GR-1 showed that the cytoplasmic chlorate reductase of P. chloritidismutans reduced only chlorate and bromate. Differences were also found in N-terminal sequences, molecular weight, and subunit composition. Metal analysis and electron paramagnetic resonance measurements showed the presence of iron and molybdenum, which are also found in other dissimilatory oxyanion reductases.