Kinetic characterisation of a dye decolourising peroxidase from Streptomyces lividans: New insight into the mechanism of anthraquinone dye decolourisation

Dye decolourising peroxidases are the most recent family of haem peroxidases to be discovered. The oxidising potential of these enzymes is driven by the formation of ferryl intermediates that enables them to oxidise synthetic dye molecules that are widely used in the textile industry. We have investigated the catalytic cycle of a dye decolourising peroxidase (DtpA) from a biotechnologically important bacterium Streptomyces lividans. Using a combination of steady-state and stopped-flow kinetic investigations, we have determined the rate constants for all steps in the catalytic cycle with a range of substrate molecules. For most substrates, the value of k cat /K m measured by steady-state kinetics is equal to the slowest step in catalysis measured by stopped-flow spectroscopy, namely the decay of the ferryl Fe IV O species (compound II) to form the ferric species. With the anthraquinone-based dye, reactive blue 19 (RB19) unusual steady-state kinetic behaviour is observed, which we propose through kinetic modelling of the catalytic cycle is due to a disproportionation mechanism of the dye. At low RB19 concentrations, the rate of disproportionation is slower than that of the rate determining step in DtpA, whereas at higher concentrations of RB19 the rate of disproportionation is faster. This mechanism obviates the need to postulate secondary sites for substrate binding on the enzyme which has been previously proposed for other dye decolourising haem peroxidases

Mapping the encounter state of a transient protein complex by PRE NMR spectroscopy

Many biomolecular interactions proceed via a short-lived encounter state, consisting of multiple, lowly-populated species invisible to most experimental techniques. Recent development of paramagnetic relaxation enhancement (PRE) nuclear magnetic resonance (NMR) spectroscopy has allowed to directly visualize such transient intermediates in a number of protein-protein and protein-DNA complexes. Here we present an analysis of the recently published PRE NMR data for a protein complex of yeast cytochrome c (Cc) and cytochrome c peroxidase (CcP). First, we describe a simple, general method to map out the spatial and temporal distributions of binding geometries constituting the Cc-CcP encounter state. We show that the spatiotemporal mapping provides a reliable estimate of the experimental coverage and, at higher coverage levels, allows to delineate the conformational space sampled by the minor species. To further refine the encounter state, we performed PRE-based ensemble simulations. The generated solutions reproduce well the experimental data and lie within the allowed regions of the encounter maps, confirming the validity of the mapping approach. The refined encounter ensembles are distributed predominantly in a region encompassing the dominant form of the complex, providing experimental proof for the results of classical theoretical simulations

The orientations of cytochrome c in the highly dynamic complex with cytochrome b5 visualized by NMR and docking using HADDOCK

The interaction of bovine microsomal ferricytochrome b5 with yeast iso-1-ferri and ferrocytochrome c has been investigated using heteronuclear NMR techniques. Chemical-shift perturbations for 1H and 15N nuclei of both cytochromes, arising from the interactions with the unlabeled partner proteins, were used for mapping the interacting surfaces on both proteins. The similarity of the binding shifts observed for oxidized and reduced cytochrome c indicates that the complex formation is not influenced by the oxidation state of the cytochrome c. Protein-protein docking simulations have been performed for the binary cytochrome b5-cytochrome c and ternary (cytochrome b5)-(cytochrome c)2 complexes using a novel HADDOCK approach. The docking procedure, which makes use of the experimental data to drive the docking, identified a range of orientations assumed by the proteins in the complex. It is demonstrated that cytochrome c uses a confined surface patch for interaction with a much more extensive surface area of cytochrome b5. Taken together, the experimental data suggest the presence of a dynamic ensemble of conformations assumed by the proteins in the complex