Global effects of the energetics of coenzyme binding: NADPH controls the protein interaction properties of human cytochrome P450 reductase
Global effects of the energetics of coenzyme binding: NADPH controls the protein interaction properties of human cytochrome P450 reductase
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DOI：
10.1021/bi052115r
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发表时间：
2006-02-07
期刊：
影响因子：
2.9
通讯作者：
Gutierrez, A
中科院分区：
文献类型：
--
作者：
Grunau, A;Paine, MJ;Gutierrez, A
用等温滴定量热法研究了辅酶与人细胞色素P450还原酶(CPR)及其FAD结合结构域的结合热力学。用离解结合常数(K-d)、结合焓(ΔH-B)、结合熵(T Delta S-B)和热容变化(Delta C-p)描述了2‘，5’ADP、NADP(+)和H(4)NaDP的结合。这一系统的方法使辅酶氧化还原状态对结合CPR的影响得以确定。辅酶-CPR复合物的识别和稳定性在很大程度上取决于与腺苷部分(K-D2‘，K-5’-ADP=76 nM)的相互作用，而与烟酰胺部分的氧化还原状态无关。2‘，5’-ADP(-210cal mol(-1)K-1)、NADP(+)(-230cal mol(-1)K-1)和H(4)NADP(-220cal mol(-1)K-1)的热容变化(Delta C-p)值相似，表明烟酰胺部分对结合表面没有显著贡献。辅酶与CPR结合的化学计量比为1：1。这一结果验证了最近提出的一位点动力学模型[Daff，S.(2004)BioChemical 43,3929-3932]，而不是我们以前提出的两位点模型[Gutierrez，A.，Lian，L.-Y.，Wolf，C.R.，Srutton，N.S.，and Roberts，C.G.K.(2001)BioChemical 40,1964-1975]。将2‘，5’-腺苷二磷酸与CPR结合(TASB=-13400+/-200calm ol(-1)，35℃)与同一配体与分离的FAD结合结构域(Tasb=-11200+/-300calm ol-1，35℃)结合进行的量热研究表明，在存在FMN结合结构域的情况下，2‘，5’-ADP结合时蛋白质可访问的构象亚态的数量增加。在研究的温度范围内(5-35摄氏度)，这种模式一直被观察到。辅酶结合能对CPR结构域动力学的贡献与先前温度跳跃研究的结论一致[Gutierrez，A.，Paine，M.，Wolf，C.R.，Srutton，N.S.，和Roberts，G.C.K.(2002)BioChemical 41,4626-4637]。量热法和停流分光光度法相结合的动力学实验表明，辅酶结合能和扩散能之间的这种联系。结构域运动直接影响CPR对细胞色素c的分子识别。CPR(k(Max)=15 S(-1)，K-d=37muM)对细胞色素c的一次周转减少是通过配体诱导的运动与辅酶结合的，使FMN结合域能够克服动力学上的非生产性构象。这是值得注意的，因为FMN结合结构域不直接参与辅酶结合，NADP(H)结合部位完全包含在FAD结合结构域中。连续的快速混合测量表明，利用辅酶结合能形成动态产生的CPR-细胞色素c复合体是一个高度同步的事件。这种有效构象(tau(50))衰变的半衰期仅为330+/-70ms。根据这些发现讨论了以前提出的结构和动力学模型。
The thermodynamics of coenzyme binding to human cytochrome P450 reductase (CPR) and its isolated FAD-binding domain have been studied by isothermal titration calorimetry. Binding of 2',5'ADP, NADP(+), and H(4)NADP, an isosteric NADPH analogue, is described in terms of the dissociation binding constant (K-d), the enthalpy (Delta H-B) and entropy (T Delta S-B) of binding, and the heat capacity change (Delta C-p). This systematic approach allowed the effect of coenzyme redox state on binding to CPR to be determined. The recognition and stability of the coenzyme-CPR complex are largely determined by interaction with the adenosine moiety (K-d2',K-5'-ADP = 76 nM), regardless of the redox state of the nicotinamide moiety. Similar heat capacity change (Delta C-p) values for 2',5'-ADP (-210 cal mol(-1) K-1), NADP(+) (-230 cal mol(-1) K-1), and H(4)NADP (-220 cal mol(-1) K-1) indicate no significant contribution from the nicotinamide moiety to the binding interaction surface. The coenzyme binding stoichiometry to CPR is 1:1. This result validates a recently proposed one-site kinetic model [Daff, S. (2004) Biochemistry 43, 3929-3932] as opposed to a two-site model previously suggested by us [Gutierrez, A., Lian, L.-Y., Wolf, C. R., Scrutton, N. S., and Roberts, C. G. K. (2001) Biochemistry 40, 1964-1975]. Calorimetric studies in which binding of 2',5'-ADP to CPR (TASB = - 13400 +/- 200 cal mol(-1), 35 degrees C) was compared with binding of the same ligand to the isolated FAD-binding domain (TASB = -11200 +/- 300 cal mol-1, 35 degrees C indicate that the number of accessible conformational substates of the protein increases upon 2',5'ADP binding in the presence of the FMN-binding domain. This pattern was consistently observed along the temperature range that was studied (5-35 degrees C). This contribution of coenzyme binding energy to domain dynamics in CPR agrees with conclusions from previous temperature-jump studies [Gutierrez, A., Paine, M., Wolf, C. R., Scrutton, N. S., and Roberts, G. C. K. (2002) Biochemistry 41, 4626-4637]. A combination of calorimetry and stopped-flow spectrophotometry kinetics experiments showed that this linkage between coenzyme binding energetics and diffusional. domain motion impinges directly on the molecular recognition of cytochrome c by CPR. Single-turnover reduction of cytochrome c by CPR (k(max) = 15 s(-1), K-d = 37 mu M) is critically coupled to coenzyme binding through ligand-induced motions that enable the FMN-binding domain to overcome a kinetically unproductive conformation. This is remarkable since the FMN-binding domain is not directly involved in coenzyme binding, the NADP(H) binding site being fully contained in the FAD-binding domain. Sequential rapid mixing measurements indicate that harnessing of coenzyme binding energy to the formation of a kinetically productive CPR-cytochrome c complex is a highly synchronized event. The inferred half-time for the decay of this productive conformation (tau(50)) is 330 +/- 70 ms only. Previously proposed structural and kinetic models are discussed in light of these findings.