Studies on the interaction of riboflavin 5'-phosphate with proteins with special attention to bacterial bioluminescence

The central theme of this thesis is the interaction of FMN with proteins. For one of the proteins studied, the enzyme luciferase from bacteria, further investigations were done on the process of light emission.In chapter 2 and 3 studies are reported on the binding of FMN with relatively simple proteins, the flavodoxins. Flavodoxins were chosen, because they are small proteins with a molecular weight not higher than 23 000. They contain only one equivalent of FMN and consist of only one polypeptide chain. No other prosthetic group is known for the flavodoxins.I n addition to this they can be obtained in high yields from bacterial cultures. These features make the flavodoxins excellent objects for studies on the binding of flavins to proteins.In chapter 2 studies on the binding of FMN by apoflavodoxin from Peptostreptococcus elsdenii are reported. Conclusions were drawn from the dependence on the pH and the NaCl concentration. The rate constant of dissociation depends on the pH, even when extrapolated to zero ionic strength. The titration curve of this rate constant can be explained, by the assumption of the involvment of two protonations, that act highly cooperatively. It should be realised that these protonations do not influence the rate constant of association. But once the complex is formed, the chance of falling apart, if these sites are protonated, is around 6 times higher than without these sites protonated. Furthermore, it was found that the calculated rate constant of association, when extrapolated to zero ionic strength is independent on the pH. At increasing ionic strength this rate constant of association will change. Depending on the pH 1 value, this change is a decrease or an increase in value. At a pH of 3.8 there is almost no change with increasing ionic strength, but above this pH value the rate constant increases, while below this value it decreases with increasing ionic strength. This explains why the combination of a high salt concentration and a low pH is a very effective way of removing the FMN from flavodoxins. This finding might possibly be extrapolated to other flavoproteins as well. By interpreting the results in terms of the Brönsted theory, a net positive charge between 11 and 12 is found on the apoenzyme at low pH. This finding is in agreement with the number of basic amino acid residues in the polypeptide chain.A series of flavin analogues were synthesised and the kinetic parameters of the interaction with Azotobacter vinelandii apoflavodoxin investigated, These studies are presented in chapter 3. Use was made of a fast kinetic method, the temperature jump relaxation technique. The resolution time of the instrument employed is 11 microseconds. All complexes studied revealed only one relaxation process, indicating that within the time limits studied (11 micro seconds - ca. 10 seconds), the association of the flavin and the apoenzyme is a one-step process. This finding is in contrast with an earlier publication by other authors, who detected two relaxation processes. It is shown that the earlier published traces are instrumental artifacts.In chapter 4 the interaction of FMN with an intermediate in the in vitro bacterial bioluminescence reaction is described. The so called "longlived lntermediate", which has been suggested to be an FMN flavoprotein, hao been separated into an apoprotein and free FMN. Because of the high quantum yields of light with :respect to FMN, measured upon reaction of the apoprotein with aldehyde, in vitro bacterial bioluminescence appears to be a sensitised reaction. At a first consideration only FMN could be a likely candidate as a sensitising agent.However, in chapter 5 it is shown that a novel protein, isolated from the bacteria itself will definitely sensitise the in vitro bacterial bioluminescence reaction. This novel protein (BFP) is efficiently fluorescent (quantum yield of fluorescence 0.45) and has an emission maximum at 476 nm. As a result of these observations, it is called the blue fluoreseence protein. By diluting this protein to a concentration of around 1 μM, a spectral shift of the emission maximum is observed. Actually the fluorescence emission spectrum of the protein changes from a spectrum identical to the In vivo bacterial bioluminescence into an emission spectrum identical to the in vitro bacterial bioluminescence emission. Although this means that the emission of this protein could account for both the in vivo and the in vitro emission spectra, it should be mentioned that investigations learned that the chromophore of this protein is not a product of the in vitro reaction. This blue fluorescence protein is the only one of all the emitters proposed so far, that simulates the bluest of the bacterial emissions exactly. Furthermore, the addition of the blue fluorescence protein to the in vitro reaction affects the light emission kinetics it acts as a catalyst), increases the light yield and induces a shift to shorter wavelengths in the bioluminescence emission. Together with the fact that the protein is isolated from the bacteria themselves, these features are strong evidence that this protein is the in vivo emitter.In chapter 6 the purification procedure of the BFP is given in more detail. Furthermore it is shown that it can be isolated from at least two of the four common species of marine bioluminescent bacteria. This suggests that all the bacteria emit their light via the same kind of chemical mechanism. Although the proteins from these two species of bacteria appear to have similar molecular weights, they differ in that the protein from P.fischeri is more tightly associated with the luciferase during the purification procedure than the one from P.phosphoreum and also that its fluorescence excitation maximum is shifted about 10 nm to shorter wavelength. Further investigation should be done in order to learn what the chemical nature of the fluorophore is.