title: Characterisation of the paramagnetic [2Fe-2S]+ centre in palustrisredoxin-B (PuxB) from Rhodopseudomonas palustris CGA009: g-matrix determination and spin coupling analysis.
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. (1)H hyperfine couplings were measured with W-band Davies ENDOR and X-band HYSCORE spectroscopy and fitted to a model in which (1)H 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 (1)H hyperfine coupling was measured using a variable mixing time ENDOR experiment to confirm the assignment of the Fe(3+) and Fe(2+) ions. For the anti-ferromagnetically coupled cluster the magnetic moment is described in terms of spin projection factors, and our analysis yields values of K(exp)(A) = +2.33 to +1.85 (ferric site), and K(exp)(B) = -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 ) 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.
authors.affiliation: Department of Chemistry, Centre for Advanced Electron Spin Resonance, University of Oxford, South Parks Road, Oxford OX1 3QR, UK.
authors.forename: Joseph A B
authors.initials: JA
authors.lastname: Abdalla
authors.forename: Alice M
authors.initials: AM
authors.lastname: Bowen
authors.forename: Stephen G
authors.initials: SG
authors.lastname: Bell
authors.forename: Luet L
authors.initials: LL
authors.lastname: Wong
authors.forename: Christiane R
authors.initials: CR
authors.lastname: Timmel
authors.forename: Jeffrey
authors.initials: J
authors.lastname: Harmer
mesh_terms: D018360:Crystallography, X-Ray; D004578:Electron Spin Resonance Spectroscopy; D005288:Ferredoxins / Q000737:chemistry*; D007501:Iron / Q000737:chemistry*; D060328:Magnetic Phenomena; D008958:Models, Molecular; D011487:Protein Conformation; D012241:Rhodopseudomonas; D013455:Sulfur / Q000737:chemistry*
chemical_list: D005288:Ferredoxins; D013455:Sulfur; D007501:Iron