Leenders R, Van Hoek A, Van Iersel M, Veeger C, Visser A J
Department of Biochemistry, Agricultural University, Wageningen, The Netherlands.
Eur J Biochem. 1993 Dec 15;218(3):977-84. doi: 10.1111/j.1432-1033.1993.tb18456.x.
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.
对从厌氧细菌拜氏梭菌中分离出的氧化型黄素氧还蛋白中黄素的时间分辨荧光特性进行了研究。使用最大熵方法分析了荧光强度衰减。结果表明，游离型和与蛋白质结合的黄素单核苷酸（FMN）在荧光行为上存在很大差异。在氧化型黄素氧还蛋白中发现了三个荧光寿命组分，其中两个在游离FMN的荧光强度衰减中不存在。主要组分分布在30皮秒，相对贡献为90%。另一个次要组分（贡献4%）分布在0.5纳秒。第三个组分分布在4.8纳秒（6%），与游离FMN荧光衰减中的主要分布一致。这些结果使我们能够确定解离常数Kd = 2.61×10⁻¹⁰ M（在20℃）。碰撞荧光猝灭实验表明，负责最长荧光寿命的黄素部分至少部分暴露于溶剂中。最短的寿命没有受到显著影响，这表明它可能源于黄素或多或少被埋在蛋白质中且碘离子无法接近的活性位点构象。使用最大熵方法对游离型和与蛋白质结合的FMN的荧光各向异性行为进行了研究和分析。发现需要过量的脱辅基黄素氧还蛋白才能检测到游离型和与蛋白质结合的FMN之间的差异。在游离FMN中检测到单一的旋转相关时间分布，而在黄素氧还蛋白中，各向异性衰减由不止一种分布组成。荧光各向异性衰减的关联分析表明，黄素氧还蛋白荧光衰减中存在的4.8纳秒荧光寿命的一部分，与溶液中游离FMN相似的旋转相关时间相关，而该寿命的另一部分与约1纳秒的较长相关时间相关。这一发现与早期研究[Barman, B. G. & Tollin, G. (1972) Biochemistry 11, 4746 - 4754]一致，在该研究中提出黄素与蛋白质的第一个结合步骤涉及磷酸基团而非FMN的另一部分。两个最短的荧光寿命，虽然不携带关于蛋白质长期旋转行为的信息，但似乎与一个与蛋白质整体翻滚相当的较长旋转相关时间相关。这些寿命组分可能源于黄素环系统或多或少固定在蛋白质基质中的复合物。
