Electron paramagnetic resonance and optical spectroscopic evidence for interaction between siroheme and Fe4S4 prosthetic groups in Escherichia coli sulfite reductase hemoprotein subunit.

The hemoprotein subunit (SiR-HP) of Escherichia coli NADPH-sulfite reductase contains one siroheme (high-spin Fe3+, D = 8 cm-1) and one oxidized Fe4S4 center per polypeptide. Christner et al. [Christner, J.A., Munck, E., Janick, P.A., & Siegel, L.M. (1981) J. Biol. Chem. 256, 2098-2101] have shown by Mossbauer spectroscopy that the two prosthetic groups of SiR-HP are magnetically exchange coupled in the oxidized enzyme, a result which indicates the presence of a chemical bridge between them. Photoreduction of SiR-HP in the presence of 5'-deazaflavin and ethylenediaminetetraacetic acid causes the enzyme to accept up to 2.0 electrons. The two reducible centers in SiR-HP are reduced independently with a midpoint potential difference of 65 mV, the siroheme being more positive. The first electron added to SiR-HP results in loss of the g = 6.63, 5.24, and 1.98 set of EPR signals due to the ferriheme and production of an EPR-silent state. The second added electron results in the parallel appearance of three distinct types of EPR signal: a novel species with g = 2.53, 2.29, and 2.07 (0.63 spin per heme); two "S = 3/2 type" species with g = 5.23, 2.80, and ca. 2.0 and g = 4.82, 3.39, and ca. 2.0 (together account for 0.16 spin per heme); and a very small amount of a "classical" reduced Fe4S4 center signal with g = 2.04, 1.93, and 1.91 (0.03 spin per heme). The temperature dependences of the "g = 2.29" and "g = 1.93" signals are similar to each other and are like those seen with other Fe4S4 center proteins. Addition of small amounts of guanidinium sulfate (0.1 M) to SiR-HP causes the spectrum of fully reduced enzyme to show primarily the S = 3/2 type species (g = 4.88, 3.31, and 2.08; 0.84 spin per heme), although the enzyme remains fully active. Optical spectral changes followed as a function of enzyme reduction show that marked changes occur in the Fe2+ siroheme optical spectrum when the Fe4S4 center becomes reduced or oxidized. These results indicate that the prosthetic groups of SiR-HP remain coupled when the enzyme is reduced. It is suggested that the novel EPR signals result from exchange interaction between S = 1 or 2 ferroheme and S = 1/2 reduced Fe4S4.

Duke Scholars

Author Lewis M. Siegel Biochemistry