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Issue 18, 2012
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Characterisation of the paramagnetic [2Fe–2S]+ centre in palustrisredoxin-B (PuxB) from Rhodopseudomonas palustris CGA009: g-matrix determination and spin coupling analysis

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Abstract

Palustrisredoxin-B (PuxB) from Rhodopseudomonas palustris (CGA009) is a [2Fe–2S] ferredoxin which is able to accept electrons from NADH via the flavin-dependent palustrisredoxin reductase (PuR); these electrons can then be transferred to the P450 enzyme (CYP199A2). This work reports on the paramagnetic state of the [2Fe–2S]+ cluster in PuxB, both alone and in the PuR–PuxB complex. Aided by the X-ray crystal structure of PuxB, the protons nearest to the reduced [2Fe–2S]+ cluster were used as magnetic probes to quantify the g-matrix orientation and anisotropic magnetic moment of the paramagnetic centre. 1H hyperfine couplings were measured with W-band Davies ENDOR and X-band HYSCORE spectroscopy and fitted to a model in which 1H dipolar couplings were calculated assuming point magnetic moments located at the Fe ions, and bridging and coordinating cysteine sulfur atoms. The absolute sign of a 1H hyperfine coupling was measured using a variable mixing time ENDOR experiment to confirm the assignment of the Fe3+ and Fe2+ ions. For the anti-ferromagnetically coupled cluster the magnetic moment is described in terms of spin projection factors, and our analysis yields values of KexpA = +2.33 to +1.85 (ferric site), and KexpB = −1.33 to −0.85 (ferrous site). These values are discussed in terms of the delocalisation of the spin density and hence the limitations of applying a local site spin coupling model to calculate the spin projection factors in a complex with considerable overlap of the α- and β-spin magnetic oribitals. The accurate description of the g-matrix orientation and magnetic moment of this [2Fe–2S]+ cluster enable it to be utilised as a paramagnetic spin probe, for example, to measure electron–electron distances. In the pdb reference frame of PuxB (code 3HUI) the g axis vector is g = [−0.6524 ± 0.0248, −0.6269 ± 0.0115, 0.4259 ± 0.0405], with the principal g-values of g = 1.9328 ± 0.0003, g = 2.0233 ± 0.0003.

Graphical abstract: Characterisation of the paramagnetic [2Fe–2S]+ centre in palustrisredoxin-B (PuxB) from Rhodopseudomonas palustris CGA009: g-matrix determination and spin coupling analysis

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Publication details

The article was received on 23 Dec 2011, accepted on 28 Feb 2012 and first published on 29 Feb 2012


Article type: Paper
DOI: 10.1039/C2CP24112A
Citation: Phys. Chem. Chem. Phys., 2012,14, 6526-6537
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    Characterisation of the paramagnetic [2Fe–2S]+ centre in palustrisredoxin-B (PuxB) from Rhodopseudomonas palustris CGA009: g-matrix determination and spin coupling analysis

    J. A. B. Abdalla, A. M. Bowen, S. G. Bell, L. L. Wong, C. R. Timmel and J. Harmer, Phys. Chem. Chem. Phys., 2012, 14, 6526
    DOI: 10.1039/C2CP24112A

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