To afford mechanistic studies in enzyme kinetics and protein folding in the microsecond time domain we have developed a continuous-flow microsecond time-scale mixing instrument with an unprecedented dead-time of 3.8 ± 0.3 μs. The instrument employs a micro-mixer with a mixing time of 2.7 μs integrated with a 30 mm long flow-cell of 109 μm optical path length constructed from two parallel sheets of silver foil; it produces ultraviolet-visible spectra that are linear in absorbance up to 3.5 with a spectral resolution of 0.4 nm. Each spectrum corresponds to a different reaction time determined by the distance from the mixer outlet, and by the fluid flow rate. The reaction progress is monitored in steps of 0.35 μs for a total duration of ~600 μs. As a proof of principle the instrument was used to study spontaneous protein refolding of pH-denatured cytochrome c. Three folding intermediates were determined: after a novel, extremely rapid initial phase with τ = 4.7 μs, presumably reflecting histidine re-binding to the iron, refolding proceeds with time constants of 83 μs and 345 μs to a coordinatively saturated low-spin iron form in quasi steady state. The time-resolution specifications of our spectrometer for the first time open up the general possibility for comparison of real data and molecular dynamics calculations of biomacromolecules on overlapping time scales. ... Read more

Detailed understanding of chemical and enzyme catalysis constitutes a main focus of current biochemical research. Fundamental insight in how (bio)catalysts function, requires knowledge of their three dimensional structure and a wide range of time resolved experiments that monitor the reaction progress. The ultimate aim is the determination of the molecular structure of transition and transient states during the chemical bond-breaking and bond-making step that occurs as part of the overall reaction. Chemists claim to have observed transient or transition states with lifetimes as short as 100-500 femtoseconds. Single steps in enzyme catalysis are usually slower than this, although electron transfer and proton transfer can occur in picoseconds or nanoseconds, respectively. The movements of protein domains which are critical to drive enzyme catalysis because they directly promote the breaking and reforming of chemical bonds, occur at a longer time scale of ~0.1-1 µs. This time range can thus be regarded as the fastest in which formation of enzyme catalytic intermediates occur or protein domains can fold into the native structure of the active enzyme. To study catalytic mechanisms of enzymes and chemical reactions in detail, the reaction should be initiated so rapidly that the subsequent formation and decay of all reaction intermediates can in fact be detected. Even the fastest present-day continuous-flow mixing equipment is too slow (~45 µs) to monitor the very beginning of enzyme catalysis. In order to design a general kinetic instrument with a much shorter dead-time to mix reactants and observe the reaction progress both the mixer and observation cell need to be miniaturized to micrometer dimensions (~100 µm) while maintaining high mixing efficiency and good optical quality. This thesis deals with the design and development of a new kinetic instrument that can perform, observe and detail, on the μs time scale, the catalytic mechanism of enzymes, in particular those of the oxidoreductases. ... Read more