EPR characterization of ascorbyl and sulfur dioxide anion radicals trapped during the reaction of bovine Cytochrome c Oxidase with molecular oxygen

Yu, Michelle; Egawa, Tsuyoshi; Yeh, Syun Ru; Rousseau, Denis L.; Gerfen, Gary J.

doi:10.1016/j.jmr.2009.12.017

Cited by 16 publications

(13 citation statements)

References 56 publications

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“…The linewidth of the natural abundance ascorbyl radical is remarkably narrow, measuring 9 G peak-to-peak, yet unresolved hyperfine splittings broaden the edges of the signal. These spectra are well-simulated as described previously confirming the assignment of the ascorbyl radical [25]. …”

Section: Resultssupporting

confidence: 81%

“…A remarkable feature of the data is the persistence of the narrow radical through 6000 µs, indicating extraordinary stability. High frequency pulsed-EPR (D-band) revealed a powder pattern consistent with approximate axial symmetry that could be simulated with g x = 2.0068, g y = 2.0066, and g z = 2.0023 (Table 1) [25]. …”

Section: Resultsmentioning

confidence: 99%

“…To determine if the radical was a reaction product of dithionite, the reaction of dithionite with both redox active and non-redox active proteins (myoglobin and bovine serum albumin) was rapidly freeze quenched. The same narrow radical signal in the X-band EPR spectrum was formed [25]. …”

Section: Resultsmentioning

confidence: 99%

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Radical formation in cytochrome c oxidase

Egawa

Shinzawa‐Itoh

et al. 2011

Biochimica et Biophysica Acta (BBA) - Bioenergetics

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The formation of radicals in bovine cytochrome c oxidase (bCcO), during the O2 redox chemistry and proton translocation, is an unresolved controversial issue. To determine if radicals are formed in the catalytic reaction of bCcO under single turnover conditions, the reaction of O2 with the enzyme, reduced by either ascorbate or dithionite, was initiated in a custom-built rapid freeze quenching (RFQ) device and the products were trapped at 77 K at reaction times ranging from 50 µs to 6 ms. Additional samples were hand mixed to attain multiple turnover conditions and quenched with a reaction time of minutes. X-band (9 GHz) continuous wave electron paramagnetic resonance (CW-EPR) spectra of the reaction products revealed the formation of a narrow radical with both reductants. D-band (130 GHz) pulsed EPR spectra allowed for the determination of the g-tensor principal values and revealed that when ascorbate was used as the reductant the dominant radical species was localized on the ascorbyl moiety, and when dithionite was used as the reductant the radical was the SO2•− ion. When the contributions from the reductants are subtracted from the spectra, no evidence for a protein-based radical could be found in the reaction of O2 with reduced bCcO. As a surrogate for radicals formed on reaction intermediates, the reaction of hydrogen peroxide (H2O2) with oxidized bCcO was studied at pH 6 and pH 8 by trapping the products at 50 µs with the RFQ device to determine the initial reaction events. For comparison, radicals formed after several minutes of incubation were also examined, and X-band and D-band analysis led to the identification of radicals on Tyr-244 and Tyr-129. In the RFQ measurements, a peroxyl (R – O – O•) species was formed, presumably by the reaction between O2 and an amino acid-based radical. It is postulated that Tyr-129 may play a central role as a proton loading site during proton translocation by ejecting a proton upon formation of the radical species and then becoming reprotonated during its reduction via a chain of three water molecules originating from the region of the propionate groups of heme a3.

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Section: Resultssupporting

confidence: 81%

Section: Resultsmentioning

confidence: 99%

See 1 more Smart Citation

Radical formation in cytochrome c oxidase

Egawa

Shinzawa‐Itoh

et al. 2011

Biochimica et Biophysica Acta (BBA) - Bioenergetics

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“…34 It should be noted that we also detect by EPR the single-line spectrum of the radical anion of SO 2 in addition to 2. This impurity signal is attributable to the known equilibrium 40 of dithionite with its two dissociated SO 2 anion radicals. Since identical amounts of dithionite are added in each run, we subtracted the background signal attributable to this radical impurity by measuring the spin concentration of the dithionite in neat buffer, which accounted for ∼1.5% spin concentration.…”

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confidence: 98%

A Noncovalently Reversible Paramagnetic Switch in Water

et al. 2013

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We report an organo-paramagnetic switch consisting of a linked bis(viologen) dication diradical that can be cycled reversibly between diamagnetic and paramagnetic states via noncovalent guest-host chemistry with cucurbit [7]uril (CB[7]) in room-temperature water. Computations suggest that the nature of the interaction between the viologen cation radical units is that of a pi dimer (pimer). Molecules with switchable magnetic properties have possible applications in spintronics, data storage devices, chemical sensors, building blocks for materials with switchable bulk magnetic properties, as well as magnetic resonance probes for biological applications. DisciplinesOrganic Chemistry | Other Chemistry | Physical Chemistry CommentsReprinted (adapted) with permission from J. Am. Chem. Soc., 2013, 135 (29) O rganic molecules with magnetic properties hold promise

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“…A small radical that is inherent to the freeze-quench procedure (Fig. 3, top trace, 'MHQ-radical') and possibly due to the ascorbyl radical [32] is seen as well. There is no evidence for the formation of a Trp-radical intermediate from the strictly conserved tryptophan in the ba 3 oxidase (W229) as it has been previously observed for the cytochrome aa 3 oxidase [21,30].…”

Section: Freeze-quench Experimentsmentioning

confidence: 95%

The cytochrome ba3 oxidase from Thermus thermophilus does not generate a tryptophan radical during turnover: Implications for the mechanism of proton pumping

Paulus

Werner

Ludwig

et al. 2015

Biochimica et Biophysica Acta (BBA) - Bioenergetics

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Oxygen reduction by cytochrome ba3 oxidase from Thermus thermophilus was studied by stopped-flow and microsecond freeze-hyperquenching analyzed with UV-Vis and EPR spectroscopy. In the initial phase, the low-spin heme b560 is rapidly and almost completely oxidized (kobs>33,000s(-1)) whereas CuA remains nearly fully reduced. The internal equilibrium between CuA and heme b560 with forward and reverse rate constants of 4621s(-1) and 3466s(-1), respectively, indicates a ~7.5mV lower midpoint potential for CuA compared to heme b560. The formation of the oxidized enzyme is relatively slow (693s(-1)). In contrast to the Paracoccus denitrificans cytochrome aa3 oxidase, where in the last phase of the oxidative half cycle a radical from the strictly conserved Trp272 is formed, no radical is formed in the cytochrome ba3 oxidase. Mutation of the Trp229, the cytochrome ba3 oxidase homologue to the Trp272, did not abolish the activity, again in contrast to the Paracoccus cytochrome aa3 oxidase. Differences in the proton pumping mechanisms of Type A and Type B oxidases are discussed in view of the proposed role of the strictly conserved tryptophan residue in the mechanism of redox-linked proton pumping in Type A oxidases. In spite of the differences between the Type A and Type B oxidases, we conclude that protonation of the proton-loading site constitutes the major rate-limiting step in both catalytic cycles.

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EPR characterization of ascorbyl and sulfur dioxide anion radicals trapped during the reaction of bovine Cytochrome c Oxidase with molecular oxygen

Cited by 16 publications

References 56 publications

Radical formation in cytochrome c oxidase

Radical formation in cytochrome c oxidase

A Noncovalently Reversible Paramagnetic Switch in Water

The cytochrome ba3 oxidase from Thermus thermophilus does not generate a tryptophan radical during turnover: Implications for the mechanism of proton pumping

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