Flavin dynamics in oxidized Clostridium beijerinckii flavodoxin as assessed by time-resolved polarized fluorescence.

The time-resolved fluorescence characteristics of flavin in oxidized flavodoxin isolated from the anaerobic bacterium Clostridium beijerinckii 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 oxidized flavodoxin, two of which are not present in the fluorescence-intensity decay of free FMN. The main component is distributed at 30 ps, with relative contribution of 90%. Another minor component (4% contribution) is distributed at 0.5 ns. The third component is distributed at 4.8 ns (6%), coinciding with the main distribution present in the fluorescence decay of free FMN. The results allowed us to determine the dissociation constant, Kd = 2.61 x 10(-10) M (at 20 degrees C). Collisional fluorescence-quenching experiments revealed that the flavin moiety responsible for the longest fluorescence lifetime is, at least partially, exposed to the solvent. The shortest lifetime is not affected significantly, 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 of free and protein-bound FMN was examined and analyzed with the maximum-entropy method. It was found that an excess of apoflavodoxin is required to detect differences between free and protein-bound FMN. In free FMN one single distribution of rotational correlation times is detected, whereas in flavodoxin the anisotropy decay is composed of more than one distribution. Associative analysis of fluorescence anisotropy decays shows that part of the 4.8 ns fluorescence lifetime present in the flavodoxin fluorescence decay, is coupled to a rotational correlation 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. This finding is in accordance with earlier studies [Barman, B. G. & Tollin, G. (1972) Biochemistry 11, 4746-4754] in which it was proposed that the first binding step of the flavin to the protein involves the phosphate group rather than another part of the FMN. The two shortest fluorescence 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 overall protein tumbling. These lifetime components probably originate from a complex in which the flavin-ring system is more or less immobilized within the protein matrix.