Fig 1.
SERR spectra of co-immobilized Mhcd1 and cyt c552.
A) Ag // 6-mercapto-1-hexanol/1-hexanethiol // cyt c552 // Mhcd1 and B) Ag // 11-amino-1-undecanethiol hydrochloride/1-undecanethiol // Mhcd1 // cyt c552 constructs at different poised potentials: 300 mV (green), 200 mV (black) and 0 mV (red). Inset: component analysis of experimental spectra (black traces) in ν4 region of co-adsorbed Mhcd1 and cyt c552 measured at 200 mV; cyt c552 (red) and Mhcd1 (green) populations; overall fit (black). Solid traces designate ferric and dotted traces ferrous ν4 components. The spectra were recorded with 413 nm excitation; laser power and accumulation time were 1.5 mW and 30 s, respectively.
Fig 2.
RR and SERR spectra of cyt c552 and Mhcd1.
A) cyt c552: RR spectra of (a) ferric and (c) sodium ascorbate reduced, ferrous protein; SERR spectra of cyt c552 immobilized on 6-mercapto-1-hexanol/1-hexanethiol SAM at electrode potentials of (b) 400 mV and (d) −100 mV. B) Mhcd1: RR spectra of (a) ferric and (c) sodium ascorbate reduced, ferrous enzyme; SERR spectra of Mhcd1 on 11-amino-1-undecanethiol hydrochloride/1-undecanethiol SAM at electrode potentials of (b) 300 mV and (d) −300 mV. The spectra were recorded with 413 nm excitation; laser power and accumulation time were 2 − 3 mW and 40 s (RR) or 1.5 − 2.5 mW and 30 s (SERR).
Table 1.
Frequencies of RR and SERR marker bands of Mhcd1 and cyt c552.
Fig 3.
Potentiometric titration of immobilized cyt c552.
SERR spectra of cyt c552 immobilized on 6-mercapto-1-hexanol/1-hexanethiol SAM-coated Ag electrode recorded at electrode potentials of (a) to (d) 250, 300, 350 and 450 mV. All spectra were measured with 413 nm excitation; laser power and accumulation time were 1.5 mW and 30 s, respectively. Inset: relative concentration of ferrous protein (squares) plotted as a function of the electrode potential. The solid line represents a fit of the experimental data to the Nernst equation, yielding E°´ = 262 ± 5 mV, z = 0.70 ± 0.02.
Fig 4.
Component analysis of the ν4 region of: RR spectra of A) ferric and C) ferrous Mhcd1 and SERR spectra of Mhcd1 immobilized on a 11-amino-1-undecanethiol hydrochloride/1-undecanethiol SAM at electrode potentials of B) 300 mV and D) −300 mV; green and red solid lines represent native ferric and ferrous populations, respectively, blue line accounts for non-native populations; gray line for non-assigned bands and black line for the overall fit. Red line in panel A indicates traces of photo-reduced protein. E) SERR spectra of Mhcd1 recorded as a function of the electrode potential, from (a) to (e) −150, −50, 50, 100 and 300 mV. The spectra were recorded with 413 nm excitation; laser power and accumulation time were 3 mW and 40 s (RR) or 2.5 mW and 30 s (SERR), respectively.
Fig 5.
Redox titrations of Mhcd1 in immobilized and solution state.
A) SERR spectroelectrochemical titration of Mhcd1 adsorbed on 11-amino-1-undecanethiol hydrochloride/1-undecanethiol coated electrodes. Data points correspond to the relative intensities of ferrous (ν4 at 1362 cm-1; solid circles) and ferric (ν4 at 1372 cm-1; open circles) heme c populations, as a function of the electrode potential. Solid lines represent fits of the Nernst equation, E°´ ~ 70 mV, z = 0.44, to the experimental data points. B) RR redox titration of Mhcd1 in solution. The relative intensities of the reduced population are represented as a function of the solution potential, solid circles. The Nernst equation was fitted to the data (black line) with E°´ = 220 ± 5 mV, z = 0.90. Inset: ν4 band of RR spectra measured at solution potentials of (a) 90, (b) 180, (c) 225 and (d) 335 mV. Component spectra represent ferrous (red) and ferric (green) ν4 populations and overall fit (black). The spectra were recorded with 413 nm excitation, with 2 − 3 mW laser power and 40 s accumulation time. Note: Sample preparation for solution RR titrations was performed in anaerobic conditions (glove box). Upon each addition of the reductant, the RR cell was removed from the glove box and the spectra were measured; a fresh aliquot of protein was used for each data point.
Fig 6.
RR spectra of Mhcd1-NO adducts and Mhcd1 prior to NO binding.
(a) Ferric and (b) ferrous NO adducts of Mhcd1 measured upon addition of diethylamine NONOate; (c) ferric and (d) ferrous Mhcd1 prior to addition of NO. The spectra were measured with 413 nm excitation, 1.6 mW laser power and 40 s accumulation time. Component spectra represent ferrous population (red), 6cLS NO adduct (light blue), 5cHS NO adduct (blue), ferric population (green), non-assigned bands (gray) and overall fit (black). Inset: low frequency region of RR spectra of ferric Mhcd1-NO adduct (top trace) and ferric Mhcd1 (bottom trace). The arrow designates the (d1)Fe-NO stretching coordinate of 5c nitrosyl Mhcd1 complex.