Cryoradiolytic reduction of heme proteins: Maximizing dose-dependent yield

Denisov, Ilia G.; Victoria, Doreen; Sligar, Stephen G.

doi:10.1016/j.radphyschem.2006.03.002

Cited by 18 publications

(17 citation statements)

References 40 publications

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“…Recent studies with other heme proteins (see (27-29) and references therein) have provided evidence for spectral and/or structural changes induced by intense X-ray radiation. The chemical, spectral and structural results described here for Thermus cytochrome ba 3 can be understood in terms of the following: First , crystalline, oxidized cytochrome ba 3 can be fully reduced by a short incubation in buffered cryo-solution containing ∼10 mM Na 2 S 2 O 4 , or alternatively, crystals can be formed from reduced protein in dithionite-containing mother liquor and maintained O 2 -free.…”

Section: Discussionmentioning

confidence: 99%

Combined Microspectrophotometric and Crystallographic Examination of Chemically Reduced and X-ray Radiation-Reduced Forms of Cytochrome ba₃ Oxidase from Thermus thermophilus: Structure of the Reduced Form of the Enzyme

et al. 2009

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Three paths are described to obtain crystals of reduced (II-E4Q/I-K258R) cytochrome ba 3 , and the structures of these are reported at ∼2.8 to 3.0 Å resolution. Microspectrophotometry of single crystals of Thermus ba 3 oxidase at 100 K was used to show that crystals of the oxidized enzyme are reduced in an intense X-ray (beam line 7-1 at the Stanford Synchrotron Radiation Laboratory, U.S.A) being nearly complete in one minute. The previously reported structures of ba 3 (PDB codes 1EHK and 1XME), having a crystallographically detectable water between the Cu B and Fe a3 metals of the dinuclear center, actually represent the X-ray radiation-reduced enzyme. Dithionite reduced crystals or crystals formed from dithionite reduced enzyme revealed the absence of the above mentioned water and an increase in the Cu B -Fe a3 distance of ∼0.3 A. The new structures are discussed in terms of enzyme function. An unexpected optical absorption envelope at ∼590 nm is also reported. This spectral feature is tentatively thought to arise from a 5-coordinate, low-spin, ferrous heme-a 3 that is trapped in the frozen crystals.In respiring organisms cytochrome c oxidase catalyzes proton translocation coupled to the reduction of O 2 to water (1,2). Cytochrome ba 3 oxidase is one of two heme-copper oxidases isolated from T. thermophilus (3). It contains the dinuclear Cu A center; a six-coordinated, lowspin, (6cLS) heme-B; and a (usually) five-coordinated, high-spin (5cHS) heme-A s in close proximity to Cu B ((3,4) and references therein). The latter (heme-A s and Cu B ) constitute the bimetallic site, and it is widely agreed that dioxygen binds to this site as the first step in O 2 -reduction and proton pumping. Two crystal structures of native and recombinant, oxidized ba 3 -type cytochrome c oxidase have been solved at <2.5 Å resolution (5,6). However, the structure of reduced cytochrome ba 3 oxidase has not been reported.Oxidized and reduced forms of cytochrome c oxidase represent two important intermediates in its catalytic cycle, and one might expect differences in their structures. Particularly, the reduced enzyme should be structurally prepared to bind O 2 (7,8). However, such structures obtained so far for the A1-type (9) enzymes indicate very small, if any, changes near the redox centers (10-12). Reported structures of both redox states from the cytochrome aa 3 of Paracoccus denitrificans also revealed essentially no changes at 3.0 to 3.3 Å resolution (10), * Corresponding authors: CDS < dave@scripps.edu >, TEL 858-784-8738, FAX 858-784-2857; JAF < jafee@scripps.edu >, TEL 858-784-9235, FAX 858-784-2857; SMS TEL 650-926-3050, FAX 650-926-3292. NIH Public Access while in the case of bovine heart cytochrome c oxidase (also of the aa 3 -type, and at a higher resolution), a redox-linked conformational change was observed in a surface loop (13) not present, for example, in ba 3 .We recently reported the structure of a doubly-mutated form of cytochrome ba 3 (II-E4Q/I-K258R) that forms robust cryst...

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Section: Discussionmentioning

confidence: 99%

Combined Microspectrophotometric and Crystallographic Examination of Chemically Reduced and X-ray Radiation-Reduced Forms of Cytochrome ba₃ Oxidase from Thermus thermophilus: Structure of the Reduced Form of the Enzyme

et al. 2009

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“…A large number of published works utilized γ-irradiation from 60 Co source for cryoradiolytic reduction of cytochromes P450 and other heme proteins (59,62,63). Photons from this source, with energies 1.13 and 1.3 MeV, interact with atoms with low Z (atomic mass) mostly through inelastic Compton scattering with emission of lower energy photon, and generation of free electron and cation radical as primary products of this scattering (60).…”

Section: Introductionmentioning

confidence: 99%

“…The actual concentration of generated electrons in ice at 77 K may be several times lower because of recombination of electrons with parent radicals, and the typical doses used in cryoradiolytic reduction range from 2 to 10 Mrad. Recombination of electrons with radicals also results in a saturation behavior of cryoradiolytic reduction with increasing dose, and the yield for metalloproteins usually is not improved by prolonged irradiation with the doses higher than 4 – 6 Mrad dose (63,64). …”

Section: Introductionmentioning

confidence: 99%

Cryoradiolysis and Cryospectroscopy for Studies of Heme-Oxygen Intermediates in Cytochromes P450

Denisov

Grinkova

Sligar

2012

Methods in Molecular Biology

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Cryogenic radiolytic reduction is one of the most simple and convenient methods of generation and stabilization of reactive iron-oxygen intermediates for mechanistic studies in chemistry and biochemistry. The method is based on one-electron reduction of the precursor complex in frozen solution via exposure to the ionizing radiation at cryogenic temperatures. Such approach allows for accumulation of the fleeting reactive complexes which otherwise could not be generated at sufficient amount for structural and mechanistic studies. Application of this method allowed for characterizing of peroxoferric and hydroperoxo-ferric intermediates, which are common for the oxygen activation mechanism in cytochromes P450, heme oxygenases and nitric oxide synthases, as well as for the peroxide metabolism by peroxidases and catalases.

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“…This undesired radiation-induced change is observed for the ferric metMb state during crystallographic data collection [8] [12] [62]. Already in the 1970s Gasyna showed that ferric Mb can be one-electron-reduced by 60 Co g-irradiation in frozen solution at 77 K [63], and a more detailed study was performed in 2007 by Denisov et al [64]. The radiation-induced reduction reduces the Fe III to Fe II , but, at this low temperature, the H 2 O that ligates to the iron is unable to move away to generate the normal ferrous deoxy [65] [66].…”

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confidence: 99%

The Influence of X‐Rays on the Structural Studies of Peroxide‐Derived Myoglobin Intermediates

Hersleth

Hsiao

Ryde

et al. 2008

Chemistry & Biodiversity

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In recent years, the awareness of potential radiation damage of metal centers in protein crystals during crystallographic data collection has received increasing attention. The radiation damage can lead to radiation-induced changes and reduction of the metal sites. One of the research fields where these concerns have been comprehensively addressed is the study of the reaction intermediates of the heme peroxidase and oxygenase reaction cycles. For both the resting states and the high-valent intermediates, the X-rays used in the structure determination have given undesired side effects through radiation-induced changes to the trapped intermediates. However, X-rays have been used to generate and trap the peroxy/hydroperoxy state in crystals. In this review, the structural work and the influence of X-rays on these intermediates in myoglobin are summarized and viewed in light of analogous studies on similar intermediates in peroxidases and oxygenases.

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Cryoradiolytic reduction of heme proteins: Maximizing dose-dependent yield

Cited by 18 publications

References 40 publications

Combined Microspectrophotometric and Crystallographic Examination of Chemically Reduced and X-ray Radiation-Reduced Forms of Cytochrome ba₃ Oxidase from Thermus thermophilus: Structure of the Reduced Form of the Enzyme

Combined Microspectrophotometric and Crystallographic Examination of Chemically Reduced and X-ray Radiation-Reduced Forms of Cytochrome ba₃ Oxidase from Thermus thermophilus: Structure of the Reduced Form of the Enzyme

Cryoradiolysis and Cryospectroscopy for Studies of Heme-Oxygen Intermediates in Cytochromes P450

The Influence of X‐Rays on the Structural Studies of Peroxide‐Derived Myoglobin Intermediates

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Cryoradiolytic reduction of heme proteins: Maximizing dose-dependent yield

Cited by 18 publications

References 40 publications

Combined Microspectrophotometric and Crystallographic Examination of Chemically Reduced and X-ray Radiation-Reduced Forms of Cytochrome ba3 Oxidase from Thermus thermophilus: Structure of the Reduced Form of the Enzyme

Combined Microspectrophotometric and Crystallographic Examination of Chemically Reduced and X-ray Radiation-Reduced Forms of Cytochrome ba3 Oxidase from Thermus thermophilus: Structure of the Reduced Form of the Enzyme

Cryoradiolysis and Cryospectroscopy for Studies of Heme-Oxygen Intermediates in Cytochromes P450

The Influence of X‐Rays on the Structural Studies of Peroxide‐Derived Myoglobin Intermediates

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Combined Microspectrophotometric and Crystallographic Examination of Chemically Reduced and X-ray Radiation-Reduced Forms of Cytochrome ba₃ Oxidase from Thermus thermophilus: Structure of the Reduced Form of the Enzyme

Combined Microspectrophotometric and Crystallographic Examination of Chemically Reduced and X-ray Radiation-Reduced Forms of Cytochrome ba₃ Oxidase from Thermus thermophilus: Structure of the Reduced Form of the Enzyme