Leenders H R, Vervoort J, van Hoek A, Visser A J
Department of Biochemistry Agricultural University, Wageningen, The Netherlands.
Eur Biophys J. 1990;18(1):43-55. doi: 10.1007/BF00185419.
The time-resolved fluorescence characteristics of tryptophan in flavodoxin isolated from the sulfate-reducing bacteria Desulfovibrio vulgaris and Desulfovibrio gigas have been examined. By comparing the results of protein preparations of normal and FMN-depleted flavodoxin, radiationless energy transfer from tryptophan to FMN has been demonstrated. Since the crystal structure of the D. vulgaris flavodoxin is known, transfer rate constants from the two excited states 1La and 1Lb can be calculated for both tryptophan residues (Trp 60 and Trp 140). Residue Trp 60, which is very close to the flavin, transfers energy very rapidly to FMN, whereas the rate of energy transfer from the remote Trp 140 to FMN is much smaller. Both tryptophan residues have the indole rings oriented in such a way that transfer will preferentially take place from the 1La excited state. The fluorescence decay of all protein preparations turned out to be complex, the parameter values being dependent on the emission wavelength. Several decay curves were analyzed globally using a model in which tryptophan is involved in some nanosecond relaxation process. A relaxation time of about 2 ns was found for both D. gigas apo- and holo-flavodoxin. The fluorescence anisotropy decay of both Desulfovibrio FMN-depleted flavodoxins is exponential, whereas that of the two holoproteins is clearly non-exponential. The anisotropy decay was analyzed using the same model as that applied for fluorescence decay. The tryptophan residues turned out to be immobilized in the protein. A time constant of a few nanoseconds results from energy transfer from tryptophan to flavin, at least for D. gigas flavodoxin. The single tryptophan residue in D. gigas flavodoxin occupies a position in the polypeptide chain remote from the flavin prosthetic group. Because of the close resemblance of steady-state and time-resolved fluorescence properties of tryptophan in both flavodoxins, the center to center distance between tryptophan and FMN in D. gigas flavodoxin is probably very similar to the distance between Trp 140 and FMN in D. vulgaris flavodoxin (i.e. 20 A).
对从普通脱硫弧菌和巨大脱硫弧菌中分离出的黄素氧还蛋白中色氨酸的时间分辨荧光特性进行了研究。通过比较正常黄素氧还蛋白和黄素单核苷酸（FMN）缺失型黄素氧还蛋白的蛋白质制备结果，证实了色氨酸到FMN的无辐射能量转移。由于已知普通脱硫弧菌黄素氧还蛋白的晶体结构，因此可以计算出色氨酸残基（Trp 60和Trp 140）从两个激发态1La和1Lb的转移速率常数。非常靠近黄素的Trp 60残基将能量非常迅速地转移到FMN，而从较远的Trp 140到FMN的能量转移速率要小得多。两个色氨酸残基的吲哚环取向使得转移将优先从1La激发态发生。所有蛋白质制备物的荧光衰减结果都很复杂，参数值取决于发射波长。使用色氨酸参与某些纳秒弛豫过程的模型对几条衰减曲线进行了全局分析。发现巨大脱硫弧菌脱辅基和全黄素氧还蛋白的弛豫时间约为2纳秒。两种脱硫弧菌FMN缺失型黄素氧还蛋白的荧光各向异性衰减是指数形式的，而两种全蛋白的荧光各向异性衰减显然是非指数形式的。使用与荧光衰减相同的模型分析各向异性衰减。结果表明色氨酸残基固定在蛋白质中。至少对于巨大脱硫弧菌黄素氧还蛋白，色氨酸到黄素的能量转移产生了几个纳秒的时间常数。巨大脱硫弧菌黄素氧还蛋白中的单个色氨酸残基在多肽链中占据远离黄素辅基的位置。由于两种黄素氧还蛋白中色氨酸的稳态和时间分辨荧光特性非常相似，巨大脱硫弧菌黄素氧还蛋白中色氨酸与FMN的中心距可能与普通脱硫弧菌黄素氧还蛋白中Trp 140与FMN的距离非常相似（即20埃）。
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