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. 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 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 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.