Autocatalytic Oscillations in the Early Phase of the Photoreduced Methyl Viologen-initiated Fast Kinetic Reaction of Hydrogenase

Abstract

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 hydrogenasemethyl 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.Hydrogenases are metalloenzymes that catalyze the reaction H 2 º 2 H ϩ ϩ 2 e Ϫ . Three distinct groups of hydrogenases can be defined, depending on the metal content of the protein. The iron-only enzymes can be found in strict anaerobic bacteria and usually display high sensitivity toward oxygen. It is known that hydrogenase needs to be activated under anaerobic conditions (4 -6). During activation and the enzyme cycle, several stable intermediates have been determined by electron paramagnetic resonance (EPR) 1 and Fourier transform infrared (FTIR) spectroscopy (7,8), ranging from the fully oxidized Form A through oxygen-free Form Si ab , and from Form B to the active enzyme (Form C and Form R). These forms have not yet been satisfactorily assigned to the corresponding theoretical enzyme forms of hydrogen splitting.Although the enzymatic activity of hydrogenase is determined routinely, a number of contradictory results have been published. Despite the many features described in the hydrogenase reaction, the activity of this class of enzymes has not yet been thoroughly explained (5, 9 -12). An autocatalytic kinetic step was recently proposed in hydrogenase activation or during the hydrogenase kinetic cycle. 2,3 The ability of nitrogen laser flash-reduced methyl or benzyl viologen to initiate a redox transition of a redox protein has been demonstrated previously (14). Here we report fast kinetic measurements of the hydrogenase enzyme reaction involving the use of an excimer laser flash-reduced redox dye. The results are evaluated on the assumption of an autocatalytic reaction in the hydrogenase kinetic cycle. EXPERIMENTAL PROCEDURESStrain and Cultivation-Thiocapsa roseopersicina BBS was a kind gift from Prof. E. N. Kondratieva (Moscow State University, Moscow). The bacteria were grown under standard photosynthetic conditions as described earlier...

Theoretical Calculations on Hydrogenase Kinetics: Explanation of the Lag Phase and the Enzyme Concentration Dependence of the Activity of Hydrogenase Uptake

“…Because a lag phase dependence may be observed in both the inactive and active hydrogenase forms (9,15,17), we can conclude from theoretical simulations that the autocatalytic step is part of the enzyme cycle. This is in good agreement with the experimental findings of special patterns during the hydrogenase-uptake reaction in a thin-layer reaction chamber and of autocatalytic oscillations in the fast absorption kinetics of the methyl viologen-initiated reaction of hydrogenase (13)(14)(15).…”

“…that the autocatalytic step occurs between two different enzyme forms (13)(14)(15). The theoretical calculations provide more evidence about the relevance of the autocatalytic model and explain some of the characteristics of the hydrogenase activity.…”

“…We recently demonstrated that the hydrogenase catalytic reaction includes at least one autocatalytic step (13)(14)(15). This assumption was based on the special patterns of the hydrogenase-uptake reaction in a thin-layer reaction chamber and on the autocatalytic oscillations in the fast absorption kinetics of the methyl viologen-initiated reaction of hydrogenase.…”

Cross-crystallization method used for the crystallization and preliminary diffraction analysis of a novel di-haem cytochromec4

“…roseopersicina strain BBS was isolated from an estuary of the White Sea (Bogorov, 1974) and was a kind gift from E. N. Kondratieva (Moscow State University, Russia). The cells were grown under standard photosynthetic conditions as described previously (Bagyinka et al, 2003). Cells were harvested in the logarithmic phase of growth and were stored at 293 K.…”

An Autocatalytic Step in the Reaction Cycle of Hydrogenase from Thiocapsa roseopersicina Can Explain the Special Characteristics of the Enzyme Reaction

“…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 already been investigated in the early phase of the hydrogenase-methyl viologen reaction (17). The results were evaluated on the assumption of an autocatalytic reaction in the hydrogenase kinetic cycle.…”