FLAVIN DYNAMICS IN OXIDIZED CLOSTRIDIUM-BEIJERINCKII FLAVODOXIN AS ASSESSED BY TIME-RESOLVED POLARIZED FLUORESCENCE

The time-resolved fluorescence characteristics of flavin in oxidized f lavodoxin isolated from the anaerobic bacterium Clostridium beijerinck ii have been examined. The fluorescence intensity decays were analyzed using the maximum-entropy method. It is demonstrated that there exist large differences in fluorescence behaviour between free and protein- bound FMN. Three fluorescence lifetime components are found in oxidize d flavodoxin, two of which are not present in the fluorescence-intensi ty decay of free FMN. The main component is distributed at 30 ps, with relative contribution of 90%. Another minor component (4% contributio n) is distributed at 0.5 ns. The third component is distributed at 4.8 ns (6%), coinciding with the main distribution present in the fluores cence decay of free FMN. The results allowed us to determine the disso ciation constant, K(d) = 2.61 X 10(-10) M (at 20-degrees-C). Collision al fluorescence-quenching experiments revealed that the flavin moiety responsible for the longest fluorescence lifetime is, at least partial ly, exposed to the solvent. The shortest lifetime is not affected sign ificantly, indicating that it possibly originates from an active-site conformation in which the flavin is more or less buried in the protein and not accessible to iodide. The fluorescence anisotropy behaviour o f free and protein-bound FMN was examined and analyzed with the maximu m-entropy method. It was found that an excess of apoflavodoxin is requ ired to detect differences between free and protein-bound FMN. In free FMN one single distribution of rotational correlation times is detect ed, whereas in flavodoxin the anisotropy decay is composed of more tha n one distribution. Associative analysis of fluorescence anisotropy de cays shows that part of the 4.8 ns fluorescence lifetime present in th e flavodoxin fluorescence decay, is coupled to a rotational correlatio n time similar to that of free FMN in solution, while another part of this lifetime is coupled to a longer correlation time of about 1 ns. T his finding is in accordance with earlier studies [Barman, B. G. & Tol lin, G. (1972) Biochemistry 11, 4746-47541 in which it was proposed th at the first binding step of the flavin to the protein involves the ph osphate group rather than another part of the FMN. The two shortest fl uorescence lifetimes, which do not carry information on the long-term rotational behaviour of the protein, seem nonetheless to be associated with a longer rotational correlation time which is comparable to over all protein tumbling. These lifetime components probably originate fro m a complex in which the flavin-ring system is more or less immobilize d within the protein matrix.