Direct Electron Transfer to the Oxygenase Domain of Neuronal Nitric Oxide Synthase (NOS):  Exploring Unique Redox Properties of NOS Enzymes

Bayachou, Mekki; Boutros, Jean A.

doi:10.1021/ja0482987

Cited by 19 publications

(20 citation statements)

References 23 publications

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“…Standard characterization reveals that CYP 2B4 displays properties that are consistent with other reports of heme-thiolate proteins in surfactant films [25, 49, 50]. In particular, the CYP 2B4 Fe III/II redox potential in DDAB is shifted to positive values relative to that determined in solution [51] (−532 mV vs. SCE for substrate-free CYP 2B4 in solution, 3 see Figure S1 in the Supplementary Data).…”

Section: Resultssupporting

confidence: 82%

Electrochemistry of mammalian cytochrome P450 2B4 indicates tunable thermodynamic parameters in surfactant films

Hagen

Gillan

et al. 2013

Journal of Inorganic Biochemistry

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Electrochemical methods continue to present an attractive means for achieving in vitro biocatalysis with cytochromes P450; however fully understanding the nature of electrode-bound P450 remains elusive. Herein we report thermodynamic parameters using electrochemical analysis of full-length mammalian microsomal cytochrome P450 2B4 (CYP 2B4) in didodecyldimethylammonium bromide (DDAB) surfactant films. Electronic absorption spectra of CYP 2B4-DDAB films on silica slides reveal an absorption maximum at 418 nm, characteristic of low-spin, six-coordinate, water-ligated FeIII heme in P450. The FeIII/II and FeII/I redox couples (E1/2) of substrate-free CYP 2B4 measured by cyclic voltammetry are −0.23 V and −1.02 V (vs. SCE, or 14 mV and −776 mV vs. NHE) at 21°C. The standard heterogeneous rate constant for electron transfer from the electrode to the heme for the FeIII/II couple was estimated at 170 s−1. Experiments indicate that the system is capable of catalytic reduction of dioxygen, however substrate oxidation was not observed. From the variation of E1/2 with temperature (18 – 40 °C), we have measured entropy and enthalpy changes that accompany heme reduction, −151 J mol−1 K−1 and −46 kJ mol−1, respectfully. The corresponding entropy and enthalpy values are less for the six-coordinate low-spin, imidazole-ligated enzyme (−59 J mol−1 K−1 and −18 kJ mol−1), consistent with limited conformational changes upon reduction. These thermodynamic parameters are comparable to those measured for bacterial P450 from Bacillus megaterium (CYP BM3), confirming our prior reports that the surfactant environment exerts a strong influence on the redox properties of the heme.

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Section: Resultssupporting

confidence: 82%

Electrochemistry of mammalian cytochrome P450 2B4 indicates tunable thermodynamic parameters in surfactant films

Hagen

Gillan

et al. 2013

Journal of Inorganic Biochemistry

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“…In particular, protein-surfactant film voltammetry has become a reliable method for achieving direct ET with heme proteins [10]. Using this technique, the Fe III/II redox couple in various heme-thiolate enzymes has been investigated [11][12][13][14]. More recently, an Fe II/I couple ($À1100 mV vs Ag/AgCl) was observed, with reductive dehalogenation catalyzed by the Fe I species [15,16].…”

Section: Introductionmentioning

confidence: 99%

Electrochemical generation of a high-valent state of cytochrome P450

Udit

Hill

Gray

2006

Journal of Inorganic Biochemistry

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“…We compared the behavior of 60/40 PC/cholesterol films to PC-only films as controls. In these electrochemical measurements, we used square wave voltammetry and focused on the FeIII/FeII redox couple of the embedded protein as a current reporter [16,17]. In general, we note that the presence of cholesterol in the film is accompanied by a significant decrease in the overall redox current measured.…”

Section: Resultsmentioning

confidence: 99%

Cholesterol levels and activity of membrane bound proteins: characterization by thermal and electrochemical methods

Zanati

Mathews²,

Perera

et al. 2009

J Therm Anal Calorim

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The long-term goal of this investigation is to study the effects of increased cholesterol levels on the molecular activity of membrane-bound enzymes such as nitric oxide synthase, that are critical in the functioning of the cardiovascular system. In this particular investigation, we used differential scanning calorimetry (DSC) and dielectric thermal analysis (DETA) to study the effect of added cholesterol on melting/recrystallization and dielectric behavior, respectively, of phosphatidylcholine (PC) bilayered thin films. We also used electrochemical methods to investigate the effect of added cholesterol on the redox behavior of the oxygenase domain of nitric oxide synthase as a probe embedded in the PC films. The results show that added cholesterol in the PC films seems to depress the molecular dynamics as indicated by lowered current responses in the presence of cholesterol as well as a slight increase of the transition temperature in the overall twophase regime behavior observed in PC cholesterol films. These results are rationalized in the context of the general DSC and DETA behaviors of the PC chol films.

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Direct Electron Transfer to the Oxygenase Domain of Neuronal Nitric Oxide Synthase (NOS): Exploring Unique Redox Properties of NOS Enzymes

Cited by 19 publications

References 23 publications

Electrochemistry of mammalian cytochrome P450 2B4 indicates tunable thermodynamic parameters in surfactant films

Electrochemistry of mammalian cytochrome P450 2B4 indicates tunable thermodynamic parameters in surfactant films

Electrochemical generation of a high-valent state of cytochrome P450

Cholesterol levels and activity of membrane bound proteins: characterization by thermal and electrochemical methods

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