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

Abstract

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 fluorescenceintensity 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--'"M (at 20°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, 4746-47541 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.Flavodoxins are low-molecular-mass electron-transport proteins (14-25 kDa) which can substitute for ferredoxin in a number of reactions (Mayhew, 1971 ;Mayhew and Ludwig, 1975;Mayhew and Tollin, 1992). Flavodoxins can be isolated from some algae and several anaerobic and aerobic bacteria grown under iron-containing or iron-deficient conditions (Knight and Hardy, 1966;Benemann et al., 1969;Mayh...

Ultrafast real-time visualization of active site flexibility of flavoenzyme thymidylate synthase ThyX

“…S1C shows that the total fluorescence decreases with increasing temperature, and that this decrease is fully reversible upon cooling. The fluorescence decrease rather than increase at higher temperatures indicates that FAD is not released at high temperature (up to 70°C), as has been observed in flavodoxin (26).…”

“…In itself, nonmonoexponential decay of the excited state of quenched flavin cofactors in flavoproteins is frequent (15). Biexponential decay has been documented spanning shorter time spans (14,36,37), and in flavodoxin multiexponentiality has been associated predominantly with partial flavin dissociation from the protein (26). In blue light-sensing using FAD (BLUF) photosensor proteins (13,31,32) and in flavin reductase (3), broader and more heterogeneous lifetime distributions have been interpreted in terms of ground-state heterogeneity.…”

Effect of Inorganic Phosphate on FMN Binding and Loop Flexibility in Desulfovibrio desulfuricans Apo-flavodoxin

“…Several studies have suggested that binding is completed in one step, although other studies indicated that the mechanism of binding may be more complex. 2,[9][10][11][12] Inspection of the X-ray crystal structure of apoflavodoxin from Anabaena PCC 7119 indicates that the phosphate-binding site is well-formed while the isoalloxazine pocket is closed by one of the aromatic residues (Trp57) normally interacting with the FMN. 8 Based on this observation, it was suggested that binding of FMN initiates by insertion of the 5 0 -phosphate at the phosphate-binding site.…”

A comparative picosecond‐resolved fluorescence study of tryptophan residues in iron‐sulfur proteins

“…One complete measurement consisted of measuring the polarized (parallel and perpendicular components) fluorescence decays of the reference compound, the sample, the background and again the reference compound. The measuring system has been shown to accurately resolve fluorescence lifetimes of 30 ps [12]. All experiments were conducted at 20°C.…”