Molecular characterisation of bacterial electron transport proteins

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Multiple forms of the catalytic centre, Cu-z, in the enzyme nitrous oxide reductase from Paracoccus pantotrophus.

Nitrous oxide reductase catalyses the reduction of nitrous oxide to dinitrogen at a unique tetranuclear copper site, called Cu(Z), which has a central inorganic sulphide ligand. Limited incubation with oxygen during the preparation of nitrous oxide reductase from Paracoccus pantotrophus results in changed redox properties of the catalytic centre by comparison with anaerobic preparations. While the anaerobically purified enzyme has a catalytic centre which performs a single electron step at a midpoint potential of E(m)=+60 mV versus the standard hydrogen electrode (n=1), the altered centre shows no redox change under similar experimental conditions. Spectroscopic properties of this 'redox fixed' centre are similar to spectra of the reduced 'redox active' form of CuZ, although the positions and intensities of a number of transitions are changed in the optical spectrum. These observations are interpreted in terms of two forms of the catalytic centre, called CuZ and CuZ*. The structural relationship between these forms is unclear. EPR and magnetic circular dichroism spectra suggest that the basic Cu4S structure is common to both. Curiously, steady-state activity of the aerobic enzyme preparation is slightly increased despite the fact the catalytic centre does not undergo detectable redox changes

Assignment of haem ligands and detection of electronic absorption bands of molybdenum in the di-haem periplasmic nitrate reductase of Paracoccus pantotrophus.

AbstractThe periplasmic nitrate reductase (NAP) from Paracoccus pantotrophus is a soluble two-subunit enzyme (NapAB) that binds two c-type haems, a [4Fe–4S] cluster and a bis-molybdopterin guanine dinucleotide cofactor that catalyses the reduction of nitrate to nitrite. In the present work the NapAB complex has been studied by magneto-optical spectroscopy to probe co-ordination of both the NapB haems and the NapA active site Mo. The absorption spectrum of the NapAB complex is dominated by features from the NapB c-type cytochromes. Using a combination of electron paramagnetic resonance spectroscopy and magnetic circular dichroism it was demonstrated that both haems are low-spin with bis-histidine axial ligation. In addition, a window between 600 and 800 nm was identified in which weak absorption features that may arise from Mo could be detected. The low-temperature MCD spectrum shows oppositely signed bands in this region (peak 648 nm, trough 714 nm) which have been assigned to S-to-Mo(V) charge transfer transitions

Molecular dissection of TatC defines critical regions essential for protein transport and a TatB-TatC contact site

The twin arginine transport (Tat) system transports folded proteins across the prokaryotic cytoplasmic membrane and the plant thylakoid membrane. TatC is the largest and most conserved component of the Tat machinery. It forms a multisubunit complex with TatB and binds the signal peptides of Tat substrates. Here we have taken a random mutagenesis approach to identify substitutions in Escherichia coli TatC that inactivate protein transport. We identify 32 individual amino acid substitutions that abolish or severely compromise TatC activity. The majority of the inactivating substitutions fall within the first two periplasmic loops of TatC. These regions are predicted to have conserved secondary structure and results of extensive amino acid insertion and deletion mutagenesis are consistent with these conserved elements being essential for TatC function. Three inactivating substitutions were identified in the fifth transmembrane helix of TatC. The inactive M205R variant could be suppressed by mutations affecting amino acids in the transmembrane helix of TatB. A physical interaction between TatC helix 5 and the TatB transmembrane helix was confirmed by the formation of a site-specific disulphide bond between TatC M205C and TatB L9C variants. This is the first molecular contact site mapped to single amino acid level between these two proteins. © 2012 Blackwell Publishing Ltd