Reaction fronts of the autocatalytic hydrogenase reaction.

We have built a model to describe the hydrogenase catalyzed, autocatalytic, reversible hydrogen oxidation reaction where one of the enzyme forms is the autocatalyst. The model not only reproduces the experimentally observed front properties, but also explains the found hydrogen ion dependence. Furthermore, by linear stability analysis, two different front types are found in good agreement with the experiments.

The autocatalytic step is an integral part of the hydrogenase cycle.

We earlier proved the involvement of an autocatalytic step in the oxidation of H(2) by HynSL hydrogenase from Thiocapsa roseopersicina, and demonstrated that two enzyme forms interact in this step. Using a modified thin-layer reaction chamber which permits quantitative analysis of the concentration of the reaction product (reduced benzyl viologen) in the reaction volume during the oxidation of H(2), we now show that the steady-state concentration of the product displays a strong enzyme concentration dependence. This experimental fact can be explained only if the previously detected autocatalytic step occurs inside the catalytic enzyme-cycle and not in the enzyme activation process. Consequently, both interacting enzyme forms should participate in the catalytic cycle of the enzyme. As far as we are aware, this is the first experimental observation of such a phenomenon resulting in an apparent inhibition of the enzyme. It is additionally concluded that the interaction of the two enzyme forms should result in a conformational change in the enzyme-substrate form. This scheme is very similar to that of prion reactions. Since merely a few molecules are involved at some point of the reaction, this process is entirely stochastic in nature. We have therefore developed a stochastic calculation method, calculations with which lent support to the conclusion drawn from the experiment.

Concentration-dependent front velocity of the autocatalytic hydrogenase reaction.

HynSL hydrogenase from Thiocapsa roseopersicina was applied to catalyze the oxidation of molecular hydrogen in a new, improved, thin-layer reaction chamber. Investigation of the nature of this catalysis via the development of reduced benzyl viologen showed clearly the typical characteristics of an autocatalytic reaction: propagation of a reaction front originating from a single point, with a constant velocity of front propagation. The dependence of the reaction velocity on enzyme concentration was a power function with a positive enzyme concentration threshold, with an exponent of 0.4 +/- 0.05. This indicates that the autocatalyst is an enzyme form. The front velocity decreased on increase of the electron acceptor concentration, as a sign that the autocatalyst interacts directly with the final electron acceptor. Overall, it may be concluded that the autocatalyst is an enzyme form in which [FeS]distal is reduced. Model calculations corroborate this. Because the reduction of all [FeS] clusters would be possible in a nonautocatalytic reaction, we hypothesize a small conformational change in the enzyme, catalyzed by the autocatalyst, which removes a block in the electron flow in either [NiFe] --> [FeS]proximal or the [FeS]proximal --> [FeS]distal reaction step, or removes a block of the penetration of gaseous hydrogen from the surface to the [NiFe] cluster.

De novo sequencing of a 21-kDa cytochrome c4 from Thiocapsa roseopersicina by nanoelectrospray ionization ion-trap and Fourier-transform ion-cyclotron resonance mass spectrometry.

We have determined the primary structure of cytochrome c(4) from Thiocapsa roseopersicina by de novo protein sequencing using the 'bottom up' approach. Three different enzymes (trypsin, endoproteinase Lys-C, and endoproteinase Glu-C) were employed to prepare four different sets of proteolytic digests. The digestion strategy was designed to permit a gradual buildup of smaller peptides into larger ones that were overlapped to yield the complete protein sequence. In this way we countered the main problem: peptides larger than about 1500 Da were difficult to sequence fully by tandem mass spectrometry. Direct infusion and online liquid chromatography were used on a linear ion trap Fourier-transform ion-cyclotron resonance hybrid instrument. The high resolving power, high mass accuracy and the availability of electron capture dissociation and collision-induced dissociation were essential to achieve full sequence coverage. The software DeNovoX complemented by manual interpretation was used to generate sequence information from tandem mass spectra. The predominantly automated nature of data acquisition and handling allowed for a relatively straightforward and fast procedure, which could compete with the mainstream alternative of nucleotide sequence determination.

Oxygen and temperature-dependent structural and redox changes in a novel cytochrome c(4) from the purple sulfur photosynthetic bacterium Thiocapsa roseopersicina.

A novel cytochrome c(4), the first of this type in purple phototrophic bacteria has been discovered in Thiocapsa roseopersicina. The fact that cytochrome c(4) has been found in an anaerobic organism puts in question the up hereto suggested role of cytochromes c(4) in the aerobic respiratory metabolism. The structure of cytochrome c(4) was studied under both aerobic and anaerobic conditions, using differential scanning calorimetry and a combination of redox potentiostatic measurements with CD and UV-Vis absorption techniques. Cytochrome c(4) maintained its functional capability at high temperature (60 degrees C) if it was kept under anaerobic conditions. With increasing temperature under aerobic conditions, however, there are dramatic conformational changes in the protein and coordination changes on the iron side. Presumably oxygen binds to the iron at the position left vacant by the methionine and facilitates conformational changes with low reversibility.

Cross-crystallization method used for the crystallization and preliminary diffraction analysis of a novel di-haem cytochrome c4.

The newly discovered di-haem cytochrome c4 from the purple sulfur photosynthetic bacterium Thiocapsa roseopersicina is the first cytochrome c4 to be crystallized from an anaerobic organism. It was crystallized using the addition of metal-ion salts to the standard vapour-diffusion method. Coloured well shaped three-dimensional crystals with dimensions of approximately 0.6 x 0.05 x 0.02 mm grew within 3-4 d at pH 5 and diffracted to 1.72 angstroms without radiation damage. Cytochrome c4 crystallized in space group P4(1)2(1)2 as a primitive tetragonal system with unit-cell parameters a = b = 75.29, c = 37.12 angstroms, alpha = beta = gamma = 90 degrees.

Theoretical calculations on hydrogenase kinetics: explanation of the lag phase and the enzyme concentration dependence of the activity of hydrogenase uptake.

Two models of the hydrogenase reaction cycle were investigated by means of theoretical calculations and model simulations. The first model is the widely accepted triangular hydrogenase reaction cycle with minor modifications; the second is a modified triangular model, where we have introduced an autocatalytic step into the reaction cycle. Both models include a one-step activation reaction. The theoretical calculations and model simulations corroborate the assumed autocatalytic reaction step concluded from the experimental characteristics of the hydrogenase reaction.

An autocatalytic step in the reaction cycle of hydrogenase from Thiocapsa roseopersicina can explain the special characteristics of the enzyme reaction.

A moving front has been observed as a special pattern during the hydrogenase-catalyzed reaction of hydrogen uptake with benzyl viologen as electron acceptor in a thin-layer reaction chamber. Such fronts start spontaneously and at random times at different points of the reaction chamber; blue spheres are seen expanding at constant speed and amplitude. The number of observable starting points depends on the hydrogenase concentration. Fronts can be initiated by injecting either a small amount of completed reaction mixture or activated hydrogenase, but not by injecting a low concentration of reduced benzyl viologen. These characteristics are consistent with an autocatalytic reaction step in the enzyme reaction. The special characteristics of the hydrogen-uptake reaction in the bulk reaction (a long lag phase, and the enzyme concentration dependence of the lag phase) support the autocatalytic nature. We conclude that there is at least one autocatalytic reaction step in the hydrogenase-catalyzed reaction. The two possible autocatalytic schemes for hydrogenase are prion-type autocatalysis, in which two enzyme forms interact, and product-activation autocatalysis, where a reduced electron acceptor and an inactive enzyme form interact. The experimental results strongly support the occurrence of prion-type autocatalysis.

Autocatalytic oscillations in the early phase of the photoreduced methyl viologen-initiated fast kinetic reaction of hydrogenase.

The kinetic characteristics of the hydrogen uptake reaction of hydrogenase, obtained by conventional activity measurements, led to the proposal of an autocatalytic reaction step in the hydrogenase cycle or during the activation process. The autocatalytic behavior of an enzyme reaction may result in oscillating concentrations of enzyme intermediates and/or products contributing to the autocatalytic step. This behavior has been investigated in the early phase of the hydrogenase-methyl viologen reaction. To measure fast hydrogenase kinetics, flash-reduced methyl viologen has been used as a light-induced trigger in transient kinetic phenomena associated with intermolecular electron transfer to hydrogenase. Here we report fast kinetic measurements of the hydrogenase-methyl viologen reaction by use of the excimer laser flash-reduced redox dye. The results are evaluated on the assumption of an autocatalytic reaction in the hydrogenase kinetic cycle. The kinetic constants of the autocatalytic reaction, i.e. the methyl viologen binding to and release from hydrogenase, were determined, and limits of the kinetic constants relating to the intramolecular (intraenzyme) reactions were set.