期刊名称：Proceedings of the National Academy of Sciences
印刷版ISSN：0027-8424
电子版ISSN：1091-6490
出版年度：2022
卷号：119
期号：30
DOI：10.1073/pnas.2205664119
语种：English
出版社：The National Academy of Sciences of the United States of America
摘要：Significance
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 (
Br
2DNiR) 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.
