Investigation of NADH binding, hydride transfer, and NAD(+) dissociation during NADH oxidation by mitochondrial complex I using modified nicotinamide nucleotides.
Investigation of NADH binding, hydride transfer, and NAD(+) dissociation during NADH oxidation by mitochondrial complex I using modified nicotinamide nucleotides.
复制标题
DOI：
10.1021/bi3016873
复制
发表时间：
2013-06-11
期刊：
影响因子：
2.9
通讯作者：
Hirst J
中科院分区：
文献类型：
--
作者：
Birrell JA;Hirst J
NADH：泛醌氧化还原酶（复合物I）是一种复杂的呼吸酶，其保存来自NADH氧化的能量，与泛醌还原偶联，作为穿过线粒体内膜的质子动力。在催化过程中，NADH被黄素单核苷酸氧化后，电子转移到铁硫簇链上。或者，黄素可以被亲水性电子受体、动力学研究中的人工电子受体或氧和氧化还原循环分子再氧化以产生活性氧。在这里，我们研究了复合物I氧化NADH机制中的两个步骤。首先，NAD（H）的分子片段，作为黄素位点抑制剂或底物进行测试，揭示了腺苷部分对结合至关重要。缺乏腺苷的含烟酰胺片段不结合，并且ADP-核糖比NAD+结合更强，表明烟酰胺对结合有害。第二，从氘代烟酰胺核苷酸的主要动力学同位素效应证实，氢化物转移是从pro-S的位置，并揭示，氢化物转移，沿着NAD+解离，是部分限速。因此，过渡态能量是平衡的，使得NADH氧化中没有一个步骤是完全限速的。只有在非常低的NADH浓度下，弱的NADH结合才限制NADH：泛醌氧化还原，而在线粒体基质的高核苷酸浓度下，弱的核苷酸结合常数有助于产物解离。使用快速的核苷酸反应和核苷酸结合常数和浓度之间的平衡，复合物I结合了快速和节能的NADH氧化与最小的超氧化物产生从无核苷酸的网站。
NADH:ubiquinone oxidoreductase (complex I) is a complicated respiratory enzyme that conserves the energy from NADH oxidation, coupled to ubiquinone reduction, as a proton motive force across the mitochondrial inner membrane. During catalysis, NADH oxidation by a flavin mononucleotide is followed by electron transfer to a chain of iron–sulfur clusters. Alternatively, the flavin may be reoxidized by hydrophilic electron acceptors, by artificial electron acceptors in kinetic studies, or by oxygen and redox-cycling molecules to produce reactive oxygen species. Here, we study two steps in the mechanism of NADH oxidation by complex I. First, molecular fragments of NAD(H), tested as flavin-site inhibitors or substrates, reveal that the adenosine moiety is crucial for binding. Nicotinamide-containing fragments that lack the adenosine do not bind, and ADP-ribose binds more strongly than NAD+, suggesting that the nicotinamide is detrimental to binding. Second, the primary kinetic isotope effects from deuterated nicotinamide nucleotides confirm that hydride transfer is from the pro-S position and reveal that hydride transfer, along with NAD+ dissociation, is partially rate-limiting. Thus, the transition state energies are balanced so that no single step in NADH oxidation is completely rate-limiting. Only at very low NADH concentrations does weak NADH binding limit NADH:ubiquinone oxidoreduction, and at the high nucleotide concentrations of the mitochondrial matrix, weak nucleotide binding constants assist product dissociation. Using fast nucleotide reactions and a balance between the nucleotide binding constants and concentrations, complex I combines fast and energy-conserving NADH oxidation with minimal superoxide production from the nucleotide-free site.
登录
查看更多内容
DOI：
10.1042/bj20081386
发表时间：
2009-01-01
期刊：
The Biochemical journal
影响因子：
--
作者：
Murphy MP
通讯作者：
Murphy MP
DOI：
10.1073/pnas.0510977103
发表时间：
2006-05-16
影响因子：
11.1
作者：
Kussmaul, Lothar;Hirst, Judy
通讯作者：
Hirst, Judy
影响因子：
--
作者：
LEHNINGER, AL
通讯作者：
LEHNINGER, AL
影响因子：
2.9
作者：
Grivermikova, Vera G.;Kotlyar, Alexander B.;Andrei, D. Vinoaradov
通讯作者：
Andrei, D. Vinoaradov
影响因子：
3.5
作者：
Birrell, James A.;King, Martin S.;Hirst, Judy
通讯作者：
Hirst, Judy