Molecular mechanism of the catalytic reaction of NO reductase revealed by novel time-resolved visible/IR absorption spectrometers with microfluidic device
Molecular mechanism of the catalytic reaction of NO reductase revealed by novel time-resolved visible/IR absorption spectrometers with microfluidic device
复制标题
新型时间分辨可见光/红外吸收光谱仪微流控装置揭示NO还原酶催化反应的分子机制
DOI：
10.1016/j.bpj.2015.11.2933
复制
发表时间：
2016
影响因子：
3.4
通讯作者：
M.
中科院分区：
文献类型：
--
作者：
Kimura;T.;Takeda;H.;Ishii;S.;Tosha;T.;Shiro;Y.;Kubo;M.
Time-resolved (TR) spectroscopy plays convincing roles in clarifying the molecular mechanism of biological reactions in the atomic and electronic level. Most of the biological reactions can be triggered by the sudden changes in buffer conditions, but the time-resolution of the conventional solution-mixing technique is limited to several milliseconds and the sample consumption is enormous, resulting in the limited applications of TR spectroscopy. Here, to investigate the enzymatic reaction of a low-yield membrane protein with microsecond-resolution, novel flow-flash TR-visible/IR spectrometers were developed. Time-resolution of microseconds was achieved using caged-compounds, which release substrates upon laser flash. Combinational use of a micro-channel flow-cell and a nano-liter step-pulse syringe-pump synchronized with the microscopic laser flashes realized the spectral accumulation with the minimal sample consumption.The developed system was applied to nitric-oxide reductase (NOR), a membrane enzyme that catalyzes NO reduction (2NO+ 2H++ 2e--> N 2 O+ H 2 O) in the bacterium denitrification process. Although our X-ray crystallographic analysis has revealed the atomic structure of catalytic center consisting of heme b 3 and non-heme Fe B, the molecular mechanism of NO reduction is still controversial. This is due to the difficulties in direct observation of the transient NO-bound form, whose lifetime is shorter than 1 ms. Our newly developed TR-visible absorption spectrometer, which probed the electronic state of heme b 3, revealed that NO bound to heme b 3 within 4 μs and was reduced with a time constant of 100 μs. TR-IR measurement at 10 μs showed that another NO molecule bound to Fe B. These TR measurements revealed that each iron in the active center binds different NO molecule in the early stage of the reaction and the subsequent NN bond formation occurs in the intramolecular manner.
