Capeillère-Blandin C, Barber M J, Bray R C
Biochem J. 1986 Sep 15;238(3):745-56. doi: 10.1042/bj2380745.
A detailed study of the electron exchanges involved between FMN and haem b2 groups within flavocytochrome b2 of yeast Hansenula anomala (H-enzyme) was performed. The results were compared with those for the homologous enzyme of yeast Saccharomyces cerevisiae (Sx-enzyme) re-investigated at 5 degrees C. The mid-point reduction potentials of FMN and haem were determined by two complementary methods: potentiometric titration with substrate, L-lactate, in the presence of dye mediators with quantification of the reduced species performed by spectrophotometry at suitable wavelengths; anaerobic titration of the enzyme by its substrate by monitoring the e.p.r. signals of the semiquinone and Fe3+ species. Values of Em,7 = -19, -23 and -45 V were determined respectively from the data for the three redox systems Ho/Hr, Fo/Fsq and Fsq/Fr in the H-enzyme instead of +6, -44 and -57 mV respectively in the Sx-enzyme [Capeillère-Blandin, Bray, Iwatsubo & Labeyrie (1975) Eur. J. Biochem. 54, 549-566]. Parallel e.p.r rapid-freezing and absorbance stopped-flow studies allowed determination of the time courses of the various redox species during their reduction by L-lactate. The flavin and the haem reduction time courses were biphasic. In the initial fast phase the reduction of flavin monitored by absorbance measurements is accomplished with a rate constant kF = 360 s-1. The reduction of the haem lags the reduction of flavin with a rate constant kH = 170 s-1. The appearance of flavin free radical is slower than the reduction in flavin absorbance and occurs with a rate constant close to that of the reduction of the haem. At saturating L-lactate concentration the initial rapid phase (up to 15 ms) involved in the overall turnover can be adequately simulated with a two-step reaction scheme. The main difference between the enzymes lies especially at the level of the first step of electron exchange between bound lactate and flavin, which for the H-enzyme is no longer the rate-limiting step in the haem reduction and becomes 8-fold faster than in the Sx-enzyme. Consequently in the H-enzyme for the following step, the intramolecular transfer from flavin hydroquinone to oxidized haem, a reliable evaluation of the rate constants becomes possible. Preliminary values are k+2 = 380 s-1 and k-2 = 120 s-1 at 5 degrees C.(ABSTRACT TRUNCATED AT 400 WORDS)
对异常汉逊酵母（H-酶）的黄素细胞色素b2中FMN和血红素b2基团之间的电子交换进行了详细研究。将结果与在5℃下重新研究的酿酒酵母同源酶（Sx-酶）的结果进行了比较。FMN和血红素的中点还原电位通过两种互补方法测定：在染料介质存在下用底物L-乳酸进行电位滴定，并通过在合适波长下的分光光度法定量还原物种；通过监测半醌和Fe3+物种的电子顺磁共振信号，用其底物对酶进行厌氧滴定。分别从H-酶中三个氧化还原系统Ho/Hr、Fo/Fsq和Fsq/Fr的数据中确定Em,7值为-19、-23和-45V，而在Sx-酶中分别为+6、-44和-57mV [Capeillère-Blandin、Bray、Iwatsubo和Labeyrie（1975年），欧洲生物化学杂志54，549-566]。平行的电子顺磁共振快速冷冻和吸光度停流研究使得能够确定各种氧化还原物种在被L-乳酸还原过程中的时间进程。黄素和血红素的还原时间进程是双相的。在初始快速阶段，通过吸光度测量监测的黄素还原以速率常数kF = 360 s-1完成。血红素的还原滞后于黄素的还原，速率常数kH = 170 s-1。黄素自由基的出现比黄素吸光度的还原慢，并且以接近血红素还原的速率常数发生。在饱和L-乳酸浓度下，总周转中涉及的初始快速阶段（长达15毫秒）可以用两步反应方案进行充分模拟。两种酶之间的主要差异尤其在于结合的乳酸和黄素之间电子交换的第一步，对于H-酶来说，这不再是血红素还原中的限速步骤，并且比Sx-酶快8倍。因此，在H-酶的下一步中，从黄素对苯二酚到氧化血红素的分子内转移，可以可靠地评估速率常数。在5℃下的初步值为k+2 = 380 s-1和k-2 = 120 s-1。（摘要截断于400字）
