Raman mode‐selective spectroscopic imaging of coenzyme and enzyme redox states

Abstract

We investigate the surface-enhanced Raman scattering (SERS) spectra of flavin monoucleotide (FMN) in its different redox states and in a redox active enzyme, nitric oxide synthase. Incubated with silver nanoparticles coated with silica, spectra for oxidized and reduced FMN are obtained at different electrochemical potentials. Dominate Raman mode shifts at 1623/1610, 1567/1550, and 1502/1492 cm À1 , belonging to typical redox-sensitive region of FMN, are observed and analyzed, and they show a consistence with the results of spectral calculation by using the density function theory (DFT) method. We assign mode at 1500 cm À1 , composed of N 5 -H bending, N 1 ¼C 10a stretching and the asymmetric C 4a -N 5 -C 5a stretching, as a spectroscopic indicator for the redox states of FMN, because it shows a significant downshift (1502/1492 cm À1 ) and non-linearly correction with potentials when FMN gets reduced from its full oxidized state electrochemically. Isolated FMN domain from a wild type neuronal nitric oxide synthase enzyme (nNOS) is also studied with the same experimental approach. We have observed similar Raman mode down-shifting as that of the pure FMN, which further supports our conclusion that the mode at~1500 cm À1 indicates redox states for FMN, the coenzyme of nNOS, even in redox protein matrix.

Raman spectroscopy probing of redox states and mechanism of flavin coenzyme

“…The Raman peaks for the oxidized states are seated on the dotted red line located at 1498 cm −1 whereas the Raman peaks for reduced states are slightly off from the red dotted line and positioned at 1492 cm −1 on the blue dotted line (Figure ). On the basis of our DFT calculations, these two Raman signals are related to 1502 and 1489 cm −1 respectively, which are originated from N 5 ─H bending, N 1 ─C 10a stretching, and the asymmetric C 4a ─N 5 ─C 5a stretching . These modes are the characteristic redox sensitive modes of FMN and corelated to protonation, deprotonation, and electron transfer processes.…”

“…[10,11] The electrochemical studies show that the redox pathways of the FMN molecules in aqueous solutions are pH dependent. [12][13][14][15][16][17][18][19][20][21][22][23][24] The redox states of FMN has an important role in blue-light photo receptor, and electron transport process during ATP synthesis. During ATP synthesis, FMN couples with the series of iron-sulfur (Fe/S) clusters in the NADH dehydrogenase (complex I) to transport the electron from NADH to ubiquinone (Q) and pump the protons from the mitochondrial matrix to its intermembrane space, this is one of the important example where different redox states of FMN involve in a biological process ( Figure S1).…”

Recent advances in linear and non‐linear Raman spectroscopy. Part XI

“…This work is the first report of SERS measurement on phosgene and diphosgene at a quantitative level . Gu and Lu described Raman mode‐selective spectroscopic imaging of coenzyme and enzyme redox states of FMN nitric oxide synthase using SERS, and they demonstrated consistency with the results of spectral calculations using DFT . Hernandez et al used SERS to study the anti‐inflammatory drug piroxicam adsorbed on the surface of silver or gold colloids as nanocarrier model.…”

“…[30] Gu and Lu described Raman mode-selective spectroscopic imaging of coenzyme and enzyme redox states of FMN nitric oxide synthase using SERS, and they demonstrated consistency with the results of spectral calculations using DFT. [31] Hernandez et al used SERS to study the anti-inflammatory drug piroxicam adsorbed on the surface of silver or gold colloids as nanocarrier model. Their work permitted them to analyze SERS spectra of piroxicam adsorbed on gold or silver nanoparticles surface at several pHs (1, 2, 4, and 7), imitating the environment of the drug in the body, either in the gastrointestinal tract or in healthy and disease tissues.…”

Role of a Conserved Tyrosine Residue in the FMN–Heme Interdomain Electron Transfer in Inducible Nitric Oxide Synthase

“…, the FMNH2 form). 36 In combination with the pH dependence profile (Table 1), we conclude that eq 2 below (assigning FMNhq as FMNH2), not eq 3 (assuming FMNhq is FMNH − ), should be used as the chemical equation for the FMN–heme IET in NOS. $$false[{\text{Fe}}^{\text{II}}false]false[{\text{FMNH}}^{•}false]+{\mathrm{H}}^{+}\rightleftharpoons false[{\text{Fe}}^{\text{III}}false]false[{\text{FMNH}}_{2}false]$$ $$false[{\text{Fe}}^{\text{II}}false]false[{\text{FMNH}}^{•}false]\rightleftharpoons false[{\text{Fe}}^{\text{III}}false]false[{\text{FMNH}}^{-}false]$$…”