Electron Paramagnetic Resonance and Optical Spectroscopic Evidence for Interaction between Siroheme and Tetranuclear Iron-Sulfur Center Prosthetic Groups in Spinach Ferredoxin-Nitrite Reductase

Spinach ferredoxin-nitrite reductase (NiR) is a monomeric enzyme containing one siroheme (high-spin Fe3+) and one oxidized Fe4S4 cluster per active molecule. When NiR is photochemically reduced with ethylenediaminetetraacetate (EDTA)-deazaflavin, the free enzyme and its CN− complex display two phases of reduction by visible absorption spectroscopy. The second phase in both cases shows changes in absorption bands associated with the heme even though the heme is already in the Fe2+ state. Similarly, the CO complex of NiR, which is formed only when the heme is in the Fe2+ state, shows changes in heme absorption bands upon reduction. In the CO and CN” complexes, these spectral changes are associated with the appearance of electron paramagnetic resonance (EPR) signals of the classical “g = 1.94” type characteristic of reduced Fe4S4 clusters. The line shapes and exact g values of these EPR signals vary between the two complexes. The second phase of reduction of free NiR is associated with the appearance of three distinct EPR signals: a small g = 1.94 type signal (0.11 spin per heme); a signal of the novel type previously observed in the Escherichia coli and spinach sulfite reductases (SiRs, also siroheme-Fe4S4 enzymes) with g⊥ = 2.40 and g∥ = 2.19 (0.15 spin per heme); an “S = 3/2” type signal with g = 5.07, 2.91, and 2.09 (0.44 spin per heme), which is similar to the two S = 3/2 signals found in E. coli SiR. Fully reduced NiR in the presence of 20% dimethyl sulfoxide has greater spin concentration in its g = 2.40 (5 = 1/2) type signal (g⊥ = 2.35, = 2.12; 0.32 spin per heme) than free NiR. In the presence of 5 mM KCl, the S = 3/2 and S =1/2 signals are enhanced while the g = 1.94 type signal is diminished. After either treatment, the enzyme remains active. These data suggest the presence of a strong magnetic interaction between the siroheme and Fe4S4 centers in spinach NiR. It is suggested that the novel EPR signals of reduced free NiR arise from exchange interaction between S = 1 or 2 ferroheme and S =1/2 reduced Fe4S4. © 1983, American Chemical Society. All rights reserved.

Duke Scholars

Author Lewis M. Siegel Biochemistry