Secrets of a proton pumping machine Mitochondrial complex I serves as a primary entry point for electrons from the tricarboxylic acid cycle into the mitochondrial electron transport chain. This massive, membrane-embedded protein complex must couple quinone reduction to conformational changes across more than 150 angstroms within four separate proton pumps. Kampjut et al. determined five structures of complex I in states along the catalytic cycle, a deactive conformation, and one with the inhibitor rotenone bound. The resolution of some structures was sufficient to see water molecules and to trace putative paths for proton transfer within the proton-pumping membrane domain. The structures add valuable details that provide a basis for generating mechanistic hypotheses for this crucial complex. Science, this issue p. eabc4209 Cryo–electron microscopy structures provide insight into proton pumping in mammalian respiratory complex I. INTRODUCTION Complex I is the first and, with 45 subunits and a total mass of ~1 MDa, the most elaborate of the mitochondrial electron transfer chain enzymes. Complex I converts energy stored in chemical bonds into a proton gradient across the membrane that drives the synthesis of adenosine triphosphate (ATP), the universal energy currency of the cell. In each catalytic cycle, the transfer of two electrons from nicotinamide adenine dinucleotide (NADH) to a hydrophobic electron carrier quinone, which happens in the peripheral arm of the enzyme, is coupled to the translocation of four protons across the inner mitochondrial membrane in the membrane arm. The exact mechanism of this energy conversion currently presents an enigma because of complex I’s size and the spatial separation between the two reactions. RATIONALE To understand the coupling mechanism of complex I, we solved its cryo–electron microscopy (cryo-EM) structures in five different conditions, including the substrate- and inhibitor-bound states and during active turnover, unlocking the various conformations that the enzyme goes through during the catalytic cycle. We also improved the resolution to up to 2.3 to 2.5 Å, allowing us to directly observe water molecules critical for proton pumping. RESULTS We showed that opening and closing movements of the peripheral and membrane arms of complex I are critical for catalysis. Opening and closing is accompanied by coordinated conformational changes at the junction between the two arms, around the quinone binding cavity. These changes involve five conserved protein loops and are initiated by the reduction of quinone, the resulting negative charge in its cavity, and decylubiquinone (DQ) movement between the deep and the shallow binding sites. The bulky inhibitor rotenone also binds at these two sites and, unexpectedly, also within ND4—one of the three antiporter-like subunits. The deactive state is defined by a notable relocation of the entire ND6 transmembrane (TM) helix 4, arresting the enzyme in the open conformation. The PSST and 49-kDa subunit loops need to be ordered in the retracted state to enable quinone reduction as observed in the turnover closed class. Upon enzyme reduction with NADH in the open state, the 49-kDa loop extends into the cavity and the ND1 subunit loop flips upward, thus ejecting the reduced quinol. Conformational changes of the ND1 and ND3 loops also transmit the conformational changes in the quinone cavity to the rest of the enzyme (labeled “coupling” in the figure) by influencing the open-closed transition in the E-channel (the proton channel nearest to the quinone site). Entire TM helices of the ND1 subunit tilt upon opening, leading to a notable rotation of the ND6 TM3 helix, accompanied by the formation of the π-bulge. Crucially, the rotation of this helix controls the formation of a critical water wire, which delivers protons from the conserved glutamates in subunit ND4L to the quinone site. This key feature brings the “charge action” of the quinone reaction directly next to ND2, the first out of the three homologous antiporter-like subunits, initiating a “wave” of electrostatic interactions propagating to the distal antiporter ND5. Analysis of water networks and charge distribution in the closed and open states of complex I under turnover explains how the protons are translocated in these waves within the antiporters and how this is coordinated between the four separate proton pumps and quinone reduction. A key role in this process is played by electrostatic interactions between the conserved charged residues, forming the highly hydrated “central axis” of the membrane arm. The distribution of the observed water molecules also suggests that links to the matrix and intermembrane space (IMS) sides in the distal subunit ND5 are much more hydrated than in other antiporters, and we propose the possibility that all four protons per cycle are ejected into the IMS via this subunit, rather than one per each antiporter (dashed arrows in the figure). CONCLUSION A comparison of conformational changes induced by substrate binding, turnover, inhibition, and deactivation allowed us to propose a detailed mechanistic model of the entire catalytic cycle in mammalian complex I, which combines elements of conformational (quinone site–E-channel) and electrostatic (antiporters) coupling. A roadmap to the binding sites, proton pathways, and coupling mechanism of complex I. A cross section of the cryo-EM density of complex I during turnover reveals an intricate machinery involved in catalysis. The solid black arrows show the propagation of the conformational changes and electrostatic interactions during the catalytic cycle; the gray arrows show the proton translocation pathways, with dashed arrows indicating the less likely paths. Core subunits are colored, and supernumerary subunits are in gray. Mitochondrial complex I couples NADH:ubiquinone oxidoreduction to proton pumping by an unknown mechanism. Here, we present cryo–electron microscopy structures of ovine complex I in five different conditions, including turnover, at resolutions up to 2.3 to 2.5 angstroms. Resolved water molecules allowed us to experimentally define the proton translocation pathways. Quinone binds at three positions along the quinone cavity, as does the inhibitor rotenone that also binds within subunit ND4. Dramatic conformational changes around the quinone cavity couple the redox reaction to proton translocation during open-to-closed state transitions of the enzyme. In the induced deactive state, the open conformation is arrested by the ND6 subunit. We propose a detailed molecular coupling mechanism of complex I, which is an unexpected combination of conformational changes and electrostatic interactions.

中文翻译：

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哺乳动物呼吸复合体Ⅰ的偶联机制

质子泵机的秘密 线粒体复合物 I 是电子从三羧酸循环进入线粒体电子传递链的主要入口点。这种巨大的膜嵌入蛋白质复合物必须将醌还原与四个独立质子泵内超过 150 埃的构象变化相结合。坎普朱特等人。确定了沿催化循环状态的复合物 I 的五种结构，一种失活构象，一种与抑制剂鱼藤酮结合。某些结构的分辨率足以看到水分子并追踪质子泵膜域内质子转移的假定路径。这些结构添加了有价值的细节，为为这个关键的复合体生成机械假设提供了基础。科学，这个问题 p。eabc4209 冷冻电子显微镜结构提供了对哺乳动物呼吸复合体 I 中质子泵送的深入了解。 简介复合体 I 是第一个，具有 45 个亚基和总质量约为 1 MDa 的线粒体电子转移链酶中最复杂的。复合物 I 将储存在化学键中的能量转化为跨膜的质子梯度，从而驱动三磷酸腺苷 (ATP) 的合成，这是细胞的通用能量货币。在每个催化循环中，两个电子从烟酰胺腺嘌呤二核苷酸 (NADH) 转移到疏水性电子载体醌（发生在酶的外周臂中）与四个质子跨膜中线粒体内膜的易位相耦合手臂。由于复合物 I 的大小和两个反应之间的空间分离，这种能量转换的确切机制目前呈现出一个谜。基本原理为了了解复合物 I 的耦合机制，我们在五种不同条件下解决了其冷冻电子显微镜 (cryo-EM) 结构，包括底物和抑制剂结合状态以及在活性周转期间，解锁酶的各种构象通过催化循环。我们还将分辨率提高到 2.3 到 2.5 Å，使我们能够直接观察对质子泵浦至关重要的水分子。结果 我们发现复合物 I 的外周臂和膜臂的打开和关闭运动对于催化作用至关重要。打开和关闭伴随着两臂交界处的协调构象变化，在醌结合腔周围。这些变化涉及五个保守的蛋白质环，由醌的还原、其空腔中产生的负电荷以及深和浅结合位点之间的癸基泛醌 (DQ) 运动引发。庞大的抑制剂鱼藤酮也在这两个位点结合，而且出人意料的是，也在 ND4 内结合 - 三个反向转运蛋白样亚基之一。失活状态的定义是整个 ND6 跨膜 (TM) 螺旋 4 的显着重新定位，将酶阻止在开放构象中。PSST 和 49-kDa 亚基循环需要在收回状态下进行排序，以实现在营业额封闭类中观察到的醌减少。当酶在开放状态下用 NADH 还原时，49-kDa 环延伸到空腔中，ND1 亚基环向上翻转，从而排出还原的醌醇。ND1 和 ND3 环的构象变化也通过影响 E 通道（最靠近质子通道的质子通道）中的开闭转变，将醌腔中的构象变化传递给酶的其余部分（图中标记为“偶联”）。醌位点）。ND1 亚基的整个 TM 螺旋在打开时倾斜，导致 ND6 TM3 螺旋显着旋转，同时形成 π 凸起。至关重要的是，该螺旋的旋转控制了关键水线的形成，它将质子从亚基 ND4L 中的保守谷氨酸传递到醌位点。这一关键特征将醌反应的“电荷作用”直接带到 ND2 旁边，ND2 是三个同源反向转运蛋白样亚基中的第一个，引发静电相互作用“波”传播到远端反向转运蛋白 ND5。对翻转下复合物 I 的闭合和开放状态下的水网络和电荷分布的分析解释了质子如何在逆向转运蛋白内的这些波中易位，以及这如何在四个独立的质子泵和醌还原之间协调。在这个过程中，保守的带电残基之间的静电相互作用发挥了关键作用，形成了膜臂的高度水合“中心轴”。观察到的水分子的分布还表明，远端亚基 ND5 中与基质和膜间空间 (IMS) 侧的连接比其他逆向转运蛋白中的水化程度要高得多，我们提出每个循环的所有四个质子都被喷射到IMS 通过这个亚基，而不是每个反向转运蛋白（图中的虚线箭头）。结论 底物结合、转换、抑制和失活引起的构象变化的比较使我们能够提出哺乳动物复合物 I 中整个催化循环的详细机制模型，该模型结合了构象（醌位点-E 通道）和静电的元素。 （反转运体）偶联。复合物 I 的结合位点、质子通路和耦合机制的路线图。 周转期间复合物 I 的冷冻电镜密度的横截面揭示了涉及催化的复杂机制。黑色实线箭头表示催化循环过程中构象变化和静电相互作用的传播；灰色箭头显示质子易位途径，虚线箭头表示不太可能的路径。核心亚基是彩色的，多余的亚基是灰色的。线粒体复合物 I 通过未知机制将 NADH：泛醌氧化还原与质子泵相结合。在这里，我们展示了绵羊复合物 I 在五种不同条件下的冷冻电子显微镜结构，包括转换，分辨率高达 2.3 到 2.5 埃。解析的水分子使我们能够通过实验定义质子易位途径。醌在沿着醌腔的三个位置结合，抑制剂鱼藤酮也在亚基 ND4 内结合。在酶的开-闭状态转换期间，醌腔周围的显着构象变化将氧化还原反应与质子易位结合起来。在诱导失活状态下，开放构象被 ND6 亚基阻止。我们提出了复合物 I 的详细分子偶联机制，