Hazzard J T, McIntire W S, Tollin G
Department of Biochemistry, University of Arizona, Tucson 85721.
Biochemistry. 1991 May 7;30(18):4559-64. doi: 10.1021/bi00232a028.
Laser flash photolysis has been used to investigate the kinetics of reduction of trimethylamine dehydrogenase by substoichiometric amounts of 5-deazariboflavin semiquinone, and the subsequent intramolecular electron transfer from the FMN cofactor to the Fe4S4 center. The initial reduction event followed second-order kinetics (k = 1.0 x 10(8) M-1 s-1 at pH 7.0 and 6.4 x 10(7) M-1 s-1 at pH 8.5) and resulted in the formation of the neutral FMN semiquinone and the reduced iron-sulfur cluster (in a ratio of approximately 1:3). Following this, a slower, protein concentration independent (and thus intramolecular) electron transfer was observed corresponding to FMN semiquinone oxidation and iron-sulfur cluster reduction (k = 62 s-1 at pH 7.0 and 30 s-1 at pH 8.5). The addition of the inhibitor tetramethylammonium chloride to the reaction mixture had no effect on these kinetic properties, suggesting that this compound exerts its effect on the reduced form of the enzyme. Treatment of the enzyme with phenylhydrazine, which introduces a phenyl group at the 4a-position of the FMN cofactor, decreased both the rate constant for reduction of the protein and the extent of FMN semiquinone production, while increasing the amount of iron-sulfur center reduction, consistent with the results obtained with the native enzyme. Experiments in which the kinetics of reduction of the enzyme were determined during various stages of partial reduction were also consistent with these results, and further indicated that the FMN semiquinone form of the enzyme is more reactive toward the deazariboflavin reductant than is the oxidized FMN.(ABSTRACT TRUNCATED AT 250 WORDS)
激光闪光光解已被用于研究亚化学计量的5-脱氮核黄素半醌对三甲胺脱氢酶的还原动力学，以及随后从FMN辅因子到Fe4S4中心的分子内电子转移。初始还原事件遵循二级动力学（在pH 7.0时k = 1.0×10⁸ M⁻¹ s⁻¹，在pH 8.5时k = 6.4×10⁷ M⁻¹ s⁻¹），并导致中性FMN半醌和还原的铁硫簇的形成（比例约为1:3）。在此之后，观察到一个较慢的、与蛋白质浓度无关（因此是分子内的）电子转移，对应于FMN半醌氧化和铁硫簇还原（在pH 7.0时k = 62 s⁻¹，在pH 8.5时k = 30 s⁻¹）。向反应混合物中加入抑制剂四甲基氯化铵对这些动力学性质没有影响，表明该化合物对酶的还原形式起作用。用苯肼处理酶，在FMN辅因子的4a位引入一个苯基，降低了蛋白质还原的速率常数和FMN半醌的产生程度，同时增加了铁硫中心的还原量，这与天然酶得到的结果一致。在部分还原的不同阶段测定酶还原动力学的实验也与这些结果一致，并且进一步表明酶的FMN半醌形式比氧化的FMN对脱氮核黄素还原剂更具反应性。（摘要截短于250字）
