Cluster characterization in iron-sulfur proteins by magnetic circular dichroism.

Abstract

We report magnetic circular dichroism (MCD) spectra of 4-Fe iron-sulfur clusters in the iron-sulfur proteins Chromatium high-potential iron protein (HIPIP) show that:(i) MCD is measurable throughout the near-infrared-visible-ultraviolet spectral range (2000-300 nm) in all three accessible oxidation states (n = 1, 2, 3; henceforth referred to as C'-, C2-, and C3-).(ii) Like the absorption spectra, but unlike the natural circular dichroism (CD) spectra, the MCD spectrum is characteristic of the cluster oxidation state and insensitive to the specific protein to which the cluster is bound. Unlike the absorption spectra, but like the natural CD spectra, the MCD spectrum exhibits appreciably structured features.(iii) The MCD spectra of the 4-Fe clusters are distinguishable from those of the 2-Fe clusters [Fe2S2(SR)4]n-(n = 2, 3).(iv) MCD is usable for the differentiation of 2-Fe and 4-Fe iron-sulfur clusters in iron-sulfur proteins.MCD measurements are reported for the iron-sulfur proteins Chromatium high-potential iron protein (HIPIP), Bacillus stearothermophilus ferredoxin (Bs ferredoxin), and Clostridium pasteurianum ferredoxin (Cp ferredoxin). In HIPIP the single cluster was studied in the C'-and C2-oxidation states.In Bs ferredoxin, which contains a single cluster, and in Cp ferredoxin, which contains two clusters, the C2-and C3-oxidation states were studied. All measurements were made on aqueous solutions at room temperature; experiments in the near-infrared required substitution of 2H20 for H20. Oxidation of reduced HIPIP was carried out by using K3Fe(CN)6. Oxidized Bs and Cp ferredoxins were reduced with Na2S204. Reduced Bs and Cp ferredoxins were handled anaerobically. MCD was measured by using a Cary 61 and an infrared CD instrument described previously (4, 5) and at magnetic fields of approximately 40 kilogauss (1 G = 10-4 T). Natural CD spectra (3) In all three oxidation states (C'-, C2-, C3-) MCD is observed down to the lowest energies (below 5000 cm'1) attained.ttOther features of note are the unusual monosignate nature of all MCD and the comparable magnitude of the MCD in paramagnetic (C'-, C3-) and diamagnetic (C2-) clusters. MCD has been reported for the 2-Fe iron-sulfur proteins spinach ferredoxin, adrenodoxin, and Spirulina maxima ferredoxin (8-10). We have repeated these measurements and extended them to include near-infrared wavelengths and to putidaredoxin (3). As in the 4-Fe proteins, the MCD is not very sensitive to the specific protein but varies with cluster oxidation state. The 2-Fe cluster MCD spectra are distinguishably different from those of the 4-Fe clusters. In particular, unlike the Abbreviations: MCD, magnetic circular dichroism; CD, natural circular dichroism; HIPIP, high-potential iron protein; Bs ferredoxin, ferredoxin from Bacillus stearothermophilus; Cp ferredoxin, ferredoxin from Clostridium pasteurianum. t Permanent address:

Circular Dichroism and X-ray Spectroscopies of Azotobacter vinelandii Nitrogenase Iron Protein

“…In contrast, the one electron oxidized state of the Fe protein was found to give a well structured CD spectrum (22). This spectrum was similar in intensity, but unique in form, to the CD observed for other proteins containing [4Fe-4S] 2ϩ clusters, which are known to be very protein environment-dependent (20,24). Both enzymatically oxidized and thionine-oxidized Fe proteins gave similar CD spectra.…”

Methods to identify and characterize iron–sulfur oligopeptides in water

“…A similar, complementary technique is magnetic circular dichroism (MCD), which incorporates a magnetic field to help distinguish between overlapping electronic transitions. Despite being superior to absorption and non-magnetic circular dichroism, 73,79 MCD spectrometers are not readily available to typical laboratories.…”

Mechanical Unfolding Pathway of the High-Potential Iron–Sulfur Protein Revealed by Single-Molecule Atomic Force Microscopy: Toward a General Unfolding Mechanism for Iron–sulfur Proteins

“…The possible reason for this small difference in mechanical rupture force between the oxidized and reduced forms of cHiPIP may lie in the mixed valence nature of the [4Fe–4S] cluster. The reduced [4Fe–4S] 2+ and the oxidized [4Fe–4S] 3+ are both mixed valence clusters ([2Fe 3+ , 2Fe 2+ ] in the reduced state and [3Fe 3+ , Fe 2+ ] in the oxidized state), and the electrons are highly delocalized in the iron–sulfur center, making the four iron ions indistinguishable by spectroscopic methods . It is thus likely that the measured rupture force for cHiPIP is an average of the rupture force for both ferric– and ferrous–thiolate bonds in cHiPIP.…”

“…The reduced [4Fe−4S] 2+ and the oxidized [4Fe−4S] 3+ are both mixed valence clusters ([2Fe 3+ , 2Fe 2+ ] in the reduced state and [3Fe 3+ , Fe 2+ ] in the oxidized state), and the electrons are highly delocalized in the iron−sulfur center, making the four iron ions indistinguishable by spectroscopic methods. 50 It is thus likely that the measured rupture force for cHiPIP is an average of the rupture force for both ferric− and ferrous− thiolate bonds in cHiPIP. The one positive charge added to the cluster by oxidation will not make significant changes to the bond strength.…”