Time-resolved resonance Raman spectroscopy of cytochrome c reduced by pulse radiolysis

Cartling, Bo; Wilbrandt, R.

doi:10.1016/0005-2728(81)90210-3

Cited by 16 publications

(10 citation statements)

References 71 publications

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“…Our interpretation of the intermediate state also is consistent with the very recent observations by timeresolved RR spectroscopy of pulse-radiolytically reduced cytochrome c (26). In that investigation, the exciting dye-laser was tuned to resonance with the a-optical absorption band.…”

Section: Discussionsupporting

confidence: 91%

“…A few years ago, this technique was applied for the first time to the study of short-lived intermediates (18,19). Time-resolved RR spectroscopy has since been used to study biological processes that involve heme proteins and are initiated by laser pulses (20)(21)(22)(23)(24)(25) or, in the case of cytochrome c reduction, by pulse radiolysis (26).…”

Section: Introductionmentioning

confidence: 99%

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Continuous flow-resonance Raman spectroscopy of an intermediate redox state of cytochrome C

Förster¹,

Hester²,

Cartling³

et al. 1982

Biophysical Journal

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An intermediate redox state of cytochrome c at alkaline pH, generated upon rapid reduction by sodium dithionite, has been observed by resonance Raman (RR) spectroscopy in combination with the continuous flow technique. The RR spectrum of the intermediate state is reported for excitation both in the (alpha, beta) and the Soret optical absorption band. The spectra of the intermediate state are more like those of the stable reduced form than those of the stable oxidized form. For excitation of 514.5 nm, the most prominent indication of an intermediate state is the wave-number shift of one RR band from 1,562 cm-1 in the stable oxidized state through 1,535 cm-1 in the intermediate state to 1,544 cm-1 in the stable reduced state. For excitation at 413.1 nm, a band, present at 1,542 cm-1 in the stable reduced state but not present in the stable oxidized state, is absent in the intermediate state. We interpret the intermediate species as the state where the heme iron is reduced but the protein remains in the conformation of the oxidized state, with methionine-80 displaced as sixth ligand to the heme iron, before relaxing to the conformation of the stable reduced state, with methionine-80 returned as sixth ligand.

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Section: Discussionsupporting

confidence: 91%

Section: Introductionmentioning

confidence: 99%

Continuous flow-resonance Raman spectroscopy of an intermediate redox state of cytochrome C

Förster¹,

Hester²,

Cartling³

et al. 1982

Biophysical Journal

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“…The observed characteristics of an intermediate state at alkaline pH include those observed in our kinetic studies of the redox transition of cyt c by time-resolved RR spectroscopy (7,8). This fact indicates that the molecular nature of the intermediate state stabilized at low temperature in the present investigation is similar to that of the transient states observed during pulse radiolytic (7) or chemical (8) reduction at room temperature.…”

Section: Rr Spectral Characteristics Of the Intermediate Redox Statessupporting

confidence: 83%

“…We have recently performed time-resolved RR studies of the kinetics of the redox transition of cytochrome c (cyt c). Both the reduction by hydrated electrons geneiated by pulse radiolysis (7) and the chemical reduction by sodium dithionite in a continuous mixed flow experiment (8) were studied. It appears desirable to obtain more detailed spectral characterizations of the intermediate states by improving the resolution and signal-to-noise ratio, which can be obtained by the time-resolved studies.…”

Section: Introductionmentioning

confidence: 99%

Intermediate and stable redox states of cytochrome c studied by low temperature resonance Raman spectroscopy

Cartling¹

1983

Biophysical Journal

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Stabilized intermediate redox states of cytochrome c are generated by radiolytic reduction of initially oxidized enzyme in glass matrices at liquid nitrogen temperature. In the intermediate states the heme group is reduced by hydrated electrons, whereas the protein conformation is restrained close to its oxidized form by the low-temperature glass matrix. The intermediate and stable redox states of cytochrome c at neutral and alkaline pH are studied by low-temperature resonance Raman spectroscopy using excitations in resonance with the B (Soret) and Q1 (beta) optical transitions. The assignments of the cytochrome c resonance Raman bands are discussed. The observed spectral characteristics of the intermediate states as well as of the alkaline transition in the oxidized state are interpreted in terms of oxidation-state marker modes, spin-state marker modes, heme iron--axial ligand stretching modes, totally symmetric in-plane porphyrin modes, nontotally symmetric in-plane modes, and out-of-plane modes.

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“…21 The assignment of the transient Raman spectra revealed the electronic structure of the lowest excited triplet state in relation to the ground state of all-trans-β-carotene. This technique was first used to characterize the conformational transition in the electron transfer protein cytochrome c. 22 The delay time between electron and the laser pulse of 11 μs was long enough to permit a fast reduction step and the subsequent processes. An absorption band at 1567 cm −1 in the stable oxidized state shifted within 1 μs to 1533 cm −1 in the transient reduced state.…”

Section: Experimental Techniques 21 Principle and Methodsmentioning

confidence: 99%

Pulse Radiolysis Studies for Mechanism in Biochemical Redox Reactions

Kobayashi

2019

Chem. Rev.

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Pulse radiolysis is a powerful method for generating highly reduced or oxidized species and free radicals. Combined with fast time-resolved spectroscopic measurement, we can monitor the reactions of intermediate species on time scales ranging from picoseconds to seconds. The application of pulse radiolysis to water generates hydrated electrons (eaq –) and specific radicals, rendering this technique useful for investigating a number of biological redox processes. The first pulse radiolysis redox investigations explored in this review involved intramolecular electron transfer processes in protein with multiple electron-accepting sites. Pulse radiolysis enabled direct monitoring of the internal electron transfer rates and the distribution of electrons within proteins. Structural information from X-ray data has allowed analysis of the rate constants and their activation parameters in relation to the mechanisms with current theoretical treatments. The second set of pulse radiolysis redox investigations explored here concerned the intermediates of enzyme reactions after redox reactions. Pulse radiolysis allowed the extremely rapid donation of electrons to a redox center in a protein. It makes it possible to observe the unstable intermediates after the reduction and the following subsequent steps. For example, the intermediates generated through the one-electron reduction of oxygenated hemoproteins, such as cytochrome P450 and nitric oxide synthase, were characterized. Interestingly, ligand exchange can occur upon the reduction of heme iron, in which different amino acid residues bind to heme in the ferrous and ferric states, respectively. We directly observed the ligand-switching intermediates of bacterial CooA, a CO sensor, and bacterial iron response regulator protein. These ligand exchange processes are physiologically important for regulating the electrode potential and effective formation of superoxide anion or HO•. The third set of pulse radiolysis redox investigations explored in this review concerns free-radical processes in biological systems. Free radicals are produced in cells and organisms in a variety of processes. The cell has developed special and very effective machinery for controlling and detoxifying reactive radicals. Radiation-generated radicals allow studies of the reactions between specific radicals and solutes, often revealing the mechanisms underlying the initial and subsequent reactions. The crucial contribution was made using pulse radiolysis techniques and knowledge of the identities, properties, and reactions of radicals. These radicals include superoxide (O2 •–), nitric monoxide (NO•), ascorbate, urate, and protein radicals. This review focuses on the reactions of these radicals and their physiological functions.

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Time-resolved resonance Raman spectroscopy of cytochrome c reduced by pulse radiolysis

Cited by 16 publications

References 71 publications

Continuous flow-resonance Raman spectroscopy of an intermediate redox state of cytochrome C

Continuous flow-resonance Raman spectroscopy of an intermediate redox state of cytochrome C

Intermediate and stable redox states of cytochrome c studied by low temperature resonance Raman spectroscopy

Pulse Radiolysis Studies for Mechanism in Biochemical Redox Reactions

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