Noble M A, Munro A W, Rivers S L, Robledo L, Daff S N, Yellowlees L J, Shimizu T, Sagami I, Guillemette J G, Chapman S K
Department of Chemistry, University of Edinburgh, U.K.
Biochemistry. 1999 Dec 14;38(50):16413-8. doi: 10.1021/bi992150w.
Midpoint reduction potentials for the flavin cofactors in the reductase domain of rat neuronal nitric oxide synthase (nNOS) in calmodulin (CaM)-free and -bound forms have been determined by direct anaerobic titration. In the CaM-free form, the FMN potentials are -49 +/- 5 mV (oxidized/semiquinone) -274 +/- 5 mV (semiquinone/reduced). The corresponding FAD potentials are -232 +/- 7, and -280 +/- 6 mV. The data indicate that each flavin can exist as a blue (neutral) semiquinone. The accumulation of blue semiquinone on the FMN is considerably higher than seen on the FAD due to the much larger separation (225 mV) of its two potentials (cf. 48 mV for FAD). For the CaM-bound form of the protein, the midpoint potentials are essentially identical: there is a small alteration in the FMN oxidized/semiquinone potential (-30 +/- 4 mV); the other three potentials are unaffected. The heme midpoint potentials for nNOS [-239 mV, L-Arg-free; -220 mV, L-Arg-bound; Presta, A., Weber-Main, A. M., Stankovich, M. T., and Stuehr, D. J. (1998) J. Am. Chem. Soc. 120, 9460-9465] are poised such that electron transfer from flavin domain is thermodynamically feasible. Clearly, CaM binding is necessary in eliciting conformational changes that enhance flavin to flavin and flavin to heme electron transfers rather than causing a change in the driving force.
通过直接厌氧滴定法测定了大鼠神经元型一氧化氮合酶（nNOS）还原酶结构域中黄素辅因子在无钙调蛋白（CaM）和结合钙调蛋白形式下的中点还原电位。在无CaM形式下，FMN的电位为-49±5 mV（氧化态/半醌态）和-274±5 mV（半醌态/还原态）。相应的FAD电位为-232±7 mV和-280±6 mV。数据表明，每种黄素都可以以蓝色（中性）半醌形式存在。由于FMN的两个电位之间的分离度（225 mV）比FAD大得多（FAD为48 mV），FMN上蓝色半醌的积累量远高于FAD。对于蛋白质的CaM结合形式，中点电位基本相同：FMN氧化态/半醌态电位有小的变化（-30±4 mV）；其他三个电位不受影响。nNOS的血红素中点电位[-239 mV，无L-精氨酸；-220 mV，结合L-精氨酸；Presta，A.，Weber-Main，A.M.，Stankovich，M.T.和Stuehr，D.J.（1998）J.Am.Chem.Soc.120，9460 - 9465]处于这样的状态，使得从黄素结构域的电子转移在热力学上是可行的。显然，CaM结合对于引发构象变化是必要的，这种构象变化增强了黄素到黄素以及黄素到血红素的电子转移，而不是导致驱动力的变化。
