Metal-nitroxyl interactions.  46.  EPR spectra of low-spin iron(III) complexes of spin-labeled tetraphenylporphyrins and their implications for the interpretation of EPR spectra of spin-labeled cytochrome P450

Fielding, Lee A.; More, Kundalika M.; Eaton, Gareth R.

doi:10.1021/ja00264a010

Cited by 14 publications

(3 citation statements)

References 3 publications

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“…3 Such a weak dipolar interaction 4 The contribution to the dynamically broadened ambient temperature spin probe line width, δH (in Gauss), from distance-dependent, dipolar interaction is as follows. δH ) (5 × 10 15 )τe/R 6 , where R (in angstrom) is the iron to nitroxide distance and τe is the ferric heme electron spin relaxation time (Fielding et al, 1986). Under ambient conditions, τe has been estimated at 2 × 10 -12 s for the heme iron of low-spin ferric cytochrome c (Keller & Wu ¨thrich, 1981), and the UV-Vis spectrum of our cytochrome c indicates that the heme is low-spin ferric.…”

Section: Discussionmentioning

confidence: 99%

Kinetics and Motional Dynamics of Spin-Labeled Yeast Iso-1-cytochrome c: 1. Stopped-Flow Electron Paramagnetic Resonance as a Probe for Protein Folding/Unfolding of the C-Terminal Helix Spin-Labeled at Cysteine 102

Vaughn

Sienkiewicz

et al. 1997

Biochemistry

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The kinetics of chemically induced folding and unfolding processes in spin-labeled yeast iso-1-cytochrome c were measured by stopped-flow electron paramagnetic resonance (EPR). Stopped-flow EPR, based on a new dielectric resonator structure [Sienkiewicz, A., Qu, K., & Scholes, C. P. (1994) Rev. Sci. Instrum. 65, 68-74], gives a new temporal component to probing nanosecond molecular tumbling motions that are modulated by macromolecular processes requiring time resolution of milliseconds to seconds. The stopped-flow EPR technique presented in this work is a kinetic technique that has not been previously used with such a time resolution on spin-labeled systems, and it has the potential for application to numerous spin-labeled sites in this and other proteins. The cysteine-specific spin-label, methanethiosulfonate spin-label (MTSSL), was attached to yeast iso-1-cytochrome c at the single naturally occurring cysteine102, and the emphasis for this work was on this disulfide-attached spin-labeled prototype. This probe has the advantage of reflecting the protein tertiary fold, as shown by recent, systematic site-directed spin labeling of T4 lysozyme [Mchaourab, H. S. Lietzow, M. A., Hideg, K., & Hubbell, W. L. (1996) Biochemistry 35, 7692-7704], and protein backbone dynamics, as also shown by model peptide studies [Todd, A. P., & Millhauser, G. L. (1991) Biochemistry 30, 5515-5523]. The C-terminal cytochrome c helix where the label is attached is thought to be critical in the initial steps of protein folding and unfolding. Stopped-flow EPR resolved the monoexponential, guanidinium-induced unfolding process at pH 6.5 with an approximately 20 ms time constant; this experiment required less than 150 microL of 80 microM spin-labeled protein. We observed an approximately 50-fold decrease of this unfolding time from the 1 s range to the 20 ms time range as the guanidinium denaturant concentration was increased from 0.6 to 2.0 M. The more complex refolding kinetics of our labeled cytochrome were studied by stopped-flow EPR at pH 5.0 and 6.5. The spin probe showed a fast kinetic process compatible with the time range over which hydrogen/deuterium amide protection indicates helix formation; this process was monoexponential at pH 5.0. At pH 6.5, there was evidence of an additional slower kinetic phase resolved by stopped-flow EPR and by heme-ligation-sensitive UV-Vis that indicated a slower folding where heme misligation may be involved. Since the disulfide-attached probe has reported folding and backbone dynamics in other systems, the implication is that our kinetic experiments were directly sensing events of the C-terminal helix formation and possibly the N- and C-terminal helical interaction. The cysteine-labeled protein was also studied under equilibrium conditions to characterize probe mobility and the effect of the probe on protein thermodynamics. The difference in spin probe mobility between folded and denatured protein was marked, and in the folded protein, the motion of the probe was anisotropically restricted. The motion...

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Section: Discussionmentioning

confidence: 99%

Kinetics and Motional Dynamics of Spin-Labeled Yeast Iso-1-cytochrome c: 1. Stopped-Flow Electron Paramagnetic Resonance as a Probe for Protein Folding/Unfolding of the C-Terminal Helix Spin-Labeled at Cysteine 102

Vaughn

Sienkiewicz

et al. 1997

Biochemistry

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“…A value of <M/R> parameter depends on the T1 value of the paramagnetic centres under investigation (Kokorin & Formazyuk, 1981). Other approaches for solving this problem as well as a perfect collection of experimentally measured values of T1 are collected in Eaton & Eaton, 2001a, 2001b In case of coupling between a nitroxide radical and a paramagnetic metal complex distributed in the matrix in pairs, interesting results were presented by (Fielding et al, 1986). Low-spin Fe(II1)-tetraphenylporphyrin complexes have been modified with seven nitroxide radicals of different length: CONHR5,6, CONHCH2R5,6, OCH2CONHR6, O(CH2)2CONHR6, O(CH2)4CONHR6.…”

Section: Interaction Between Radicals and Paramagnetic Metal Ionsmentioning

confidence: 99%

“…The EPR spectra, reported by other authors, of two spin labels coordinated to ferric cytochrome P450 were analyzed with the computer programs developed for the iron porphyrin model systems. The authors showed that electron-electron exchange interaction as well as dipolar interaction must be considered in analyzing the spectra of spin-labeled porphyrin-containing bio-macromolecules (Fielding et al, 1986).…”

Section: Interaction Between Radicals and Paramagnetic Metal Ionsmentioning

confidence: 99%

Forty Years of the d1/d Parameter

Кокорин¹

2012

Nitroxides - Theory, Experiment and Applications

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ChemInform Abstract: Metal ‐ Nitroxyl Interactions. Part 46. EPR Spectra of Low‐Spin Iron(III) Complexes of Spin‐Labeled Tetraphenylporphyrins and Their Implications for the Interpretation of EPR Spectra of Spin‐Labeled Cytochrome P450.

Fielding¹,

More²,

Eaton³

1986

Chemischer Informationsdienst

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Metal-nitroxyl interactions. 46. EPR spectra of low-spin iron(III) complexes of spin-labeled tetraphenylporphyrins and their implications for the interpretation of EPR spectra of spin-labeled cytochrome P450

Abstract: The spin‐labeled iron porphyrins were prepared by reaction of Fe(II) with ortho‐substituted porphyrins followed by attachment of the spin label because attempts to react Fe(II) with spinlabeled porphyrins resulted in reduction of the nitroxyl radical.

Cited by 14 publications

References 3 publications

Kinetics and Motional Dynamics of Spin-Labeled Yeast Iso-1-cytochrome c: 1. Stopped-Flow Electron Paramagnetic Resonance as a Probe for Protein Folding/Unfolding of the C-Terminal Helix Spin-Labeled at Cysteine 102

Kinetics and Motional Dynamics of Spin-Labeled Yeast Iso-1-cytochrome c: 1. Stopped-Flow Electron Paramagnetic Resonance as a Probe for Protein Folding/Unfolding of the C-Terminal Helix Spin-Labeled at Cysteine 102

Forty Years of the d1/d Parameter

ChemInform Abstract: Metal ‐ Nitroxyl Interactions. Part 46. EPR Spectra of Low‐Spin Iron(III) Complexes of Spin‐Labeled Tetraphenylporphyrins and Their Implications for the Interpretation of EPR Spectra of Spin‐Labeled Cytochrome P450.

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