Single crystal spectroscopy and multiple structures from one crystal (MSOX) define catalysis in copper nitrite reductases.-猫眼课题宝

DOI：

10.1073/pnas.2205664119

发表时间：

2022-07-26

期刊：

Proceedings of the National Academy of Sciences of the United States of America

影响因子：

11.1

通讯作者：

中科院分区：

综合性期刊1区

文献类型：

作者：

来源链接：

https://www.ncbi.nlm.nih.gov/pmc/articles/PMC9335323/

用于收集晶体学数据的x射线可以改变包括金属蛋白在内的许多生物系统所利用的过渡金属的氧化还原态。这一缺点已被利用来驱动复杂的化学反应，需要将电子传递到活性位点，并记录催化过程中伴随的结构变化，从而提供酶促反应的实时结构电影，这是酶学家几十年来的梦想。通过将单晶技术的多结构与单晶和溶液光学光谱相结合，我们发现在最近表征的亚硝酸盐铜还原酶中，电子在接受电子的1型Cu和催化型2型Cu氧化还原中心之间的转移是门控的。这种结构/光谱结合的方法适用于许多复杂的氧化还原生物系统。许多酶利用氧化还原偶联中心进行催化，这些中心用于控制和调节催化所需的电子转移，其不及时的传递会导致无法结合底物的状态，即死端酶。亚硝酸盐铜还原酶（CuNiRs）催化亚硝酸盐还原为一氧化氮（NO），是研究质子耦合电子转移（ET）及其配合底物结合/利用等复杂过程的良好模型系统。最近，一个来自根瘤菌物种的双域CuNiR （Br2DNiR）被发现具有明显较低的酶活性，其中催化型2Cu （T2Cu）位点被两个水分子占据，需要它们的位移才能与底物亚硝酸盐结合。分离晶体和亚硝酸盐浸泡晶体的单晶光谱结合MSOX（来自一个晶体的多个结构）清楚地表明，耦合T1Cu-T2Cu氧化还原体系中的cu间ET是严重门控的。激光光解和光谱分析表明，光激发NADH到T1Cu中心的ET快速，但在没有亚硝酸盐的情况下，cu间ET很少或没有。此外，在亚硝酸盐存在的情况下，观察到T1Cu位点的不完全再氧化（转移了约20%的电子），这与从亚硝酸盐浸泡晶体中获得的MSOX薄膜的系列结构中缓慢形成NO相一致，这可能是导致该CuNiR活性较低的原因。我们的方法与研究广泛生物系统中的氧化还原反应直接相关，包括至少占所有蛋白质30%的金属蛋白。

X-rays used to collect crystallographic data can change the redox states of transition metals utilized by many biological systems including metalloproteins. This disadvantage has been harnessed to drive a complex chemical reaction requiring the delivery of an electron to the active site and recording the structural changes accompanying catalysis, providing a real-time structural movie of an enzymatic reaction, which has been a dream of enzymologists for decades. By coupling the multiple-structures from one crystal technique with single-crystal and solution optical spectroscopy, we show that the electron transfer between the electron accepting type-1 Cu and catalytic type-2 Cu redox centers is gated in a recently characterized copper nitrite reductase. This combined structural/spectroscopic approach is applicable to many complex redox biological systems. Many enzymes utilize redox-coupled centers for performing catalysis where these centers are used to control and regulate the transfer of electrons required for catalysis, whose untimely delivery can lead to a state incapable of binding the substrate, i.e., a dead-end enzyme. Copper nitrite reductases (CuNiRs), which catalyze the reduction of nitrite to nitric oxide (NO), have proven to be a good model system for studying these complex processes including proton-coupled electron transfer (ET) and their orchestration for substrate binding/utilization. Recently, a two-domain CuNiR from a Rhizobia species (Br2DNiR) has been discovered with a substantially lower enzymatic activity where the catalytic type-2 Cu (T2Cu) site is occupied by two water molecules requiring their displacement for the substrate nitrite to bind. Single crystal spectroscopy combined with MSOX (multiple structures from one crystal) for both the as-isolated and nitrite-soaked crystals clearly demonstrate that inter-Cu ET within the coupled T1Cu-T2Cu redox system is heavily gated. Laser-flash photolysis and optical spectroscopy showed rapid ET from photoexcited NADH to the T1Cu center but little or no inter-Cu ET in the absence of nitrite. Furthermore, incomplete reoxidation of the T1Cu site (∼20% electrons transferred) was observed in the presence of nitrite, consistent with a slow formation of NO species in the serial structures of the MSOX movie obtained from the nitrite-soaked crystal, which is likely to be responsible for the lower activity of this CuNiR. Our approach is of direct relevance for studying redox reactions in a wide range of biological systems including metalloproteins that make up at least 30% of all proteins.