Redox chemistry of the Schizosaccharomyces pombe ferredoxin electron-transfer domain and influence of Cys to Ser substitutions

Abstract

Schizosaccharomyces pombe (Sp) ferredoxin contains a C-terminal electron transfer protein ferredoxin domain (etp(Fd)) that is homologous to adrenodoxin. The ferredoxin has been characterized by spectroelectrochemical methods, and Mössbauer, UV-Vis and circular dichroism spectroscopies. The Mössbauer spectrum is consistent with a standard diferric [2Fe-2S](2+) cluster. While showing sequence homology to vertebrate ferredoxins, the E°' and the reduction thermodynamics for etp(Fd) (-0.392 V) are similar to plant-type ferredoxins. Relatively stable Cys to Ser derivatives were made for each of the four bound Cys residues and variations in the visible spectrum in the 380-450 nm range were observed that are characteristic of oxygen ligated clusters, including members of the [2Fe-2S] cluster IscU/ISU scaffold proteins. Circular dichroism spectra were similar and consistent with no significant structural change accompanying these mutations. All derivatives were active in an NADPH-Fd reductase cytochrome c assay. The binding affinity of Fd to the reductase was similar, however, V(max) reflecting rate limiting electron transfer was found to decrease ~13-fold. The data are consistent with relatively minor perturbations of both the electronic properties of the cluster following substitution of the Fe-bond S atom with O, and the electronic coupling of the cluster to the protein.

Fig. 1

Sequence alignment of Sp etp^Fd, human ferredoxin and bovine adrenodoxin. Residues conserved are indicated by ‘:’, the same charged residues are indicated by ‘.’ . The yellow region indicates the acidic interaction domain.

Fig. 2

Electronic spectra of S. pombe etp^Fd obtained at various applied potentials in spectroelectrochemical experiments carried out with an OTTLE cell. Spectra were recorded at 25 °C. The corresponding Nernst plots are reported in the inserts, where X stands for: [A_λox/(A_λox^Max-A_λox)], and λ_ox=415 nm.

Fig. 3

Temperature dependence of the reduction potential of S. pombe etp^Fd. The ΔS°’_rc and ΔH°’_rc values are available from the slope in (A) and (B), respectively. Dashed lines are least-squares fits to the data points.

Fig. 4

100 K Mössbauer spectrum of Sp etp^Fd in a 450 G applied field, protein was in 50 mM Tris-HCl (pH 7.5), 50 mM NaCl.

Fig. 5

(Top) UV-Vis absorption spectra of holo Sp etp^Fd (solid line) and apo Sp etp^Fd (dotted line). (Bottom) UV-Vis absorption spectra of mixture of apo and holo Cys-to-Ser Sp etp^Fd mutants. Spectra were taken in 50 mM Tris-HCl buffer, 50 mM NaCl, pH 7.5.

Fig. 6

Circular dichroism spectra of Sp etp^Fd and Hs Fd. Spectra were determined for 70 μM proteins in 10 mM potassium phosphate, pH 7.4, and the signal intensity was adjusted to the same protein concentration. Spectra in the visible and near-UV regions were recorded by using 10-mm path length cells.

Fig. 7

The plots, 1/V (nmol cyt. c reduced/min)⁻¹ versus 1/[Fd], of Cytochrome c assay. Reaction mixtures contained 80 μM cytochrome c and 200 nM Fd reductase in 1 mL of potassium phosphate buffer (10 mM, pH 7.5). The reaction was initiated by the addition of holo Fd and NADPH (400 μM) under anaerobic conditions and monitored by measuring the increase in absorbance at 550 nm from reduced cytochrome c. The values of K_m and V_max were available from the intercepts.