Molecular Mechanism of the Catalytic Reaction of no Reductase Revealed by Novel Time-Resolved Visible/IR Absorption Spectrometers with Microfluidic Device

Abstract

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- -> N2O + H2O) in the bacterium denitrification process. Although our X-ray crystallographic analysis has revealed the atomic structure of catalytic center consisting of heme b3 and non-heme FeB, 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 b3, revealed that NO bound to heme b3 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 FeB. 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 N-N bond formation occurs in the intramolecular manner.