JBC, Vol. 250, Issue 21, 8330-8336, Nov, 1975

Characterization of two soluble ferredoxins as distinct from bound iron-sulfur proteins in the photosynthetic bacterium Rhodospirillum rubrum

D. C. Yoch, D. I. Arnon and W. V. Sweeney

In an earlier investigation (Shanmugam, K. T., Buchanan, B. B., and Arnon, D. I. (1972) Biochim. Biophys. Acta 256, 477-486) the extraction of ferredoxin from Rhodospirillum rubrum cells with the aid of a detergent (Triton X-100) and acetone revealed the existence of two types of ferredoxin (I and II) and led to the conclusion that both are membrane-bound. In the present investigation, ferredoxin and acid-labile sulfur analyses of photosynthetic membranes (chromatophores) and soluble protein extracts of the photosynthetic bacteria R. rubrum and Rhodopseudomonas spheroides showed that ferredoxins I and II are primarily components of the soluble protein fraction. After their removal, washed R. rubrum chromatophores were found to contain a considerable amount of tightly bound iron-sulfur protein(s), as evidenced by acid-labile sulfur and electron paramagnetic resonance analyses. Thus, like all other photosynthetic cells examined to date, R. rubrum cells contain both soluble ferredoxins and iron-sulfur proteins tightly bound to photosynthetic membranes. The molecular weights of ferredoxins I and II from photosynthetically grown R. rubrum cells were found to be 8,800 and 14,500, respectively. Using these molecular weights, the molar extinction coefficients at 390 nm for ferredoxins I and II were determined to be 30.3 and 17.2 mM-1 CM-1, respectively. Ferredoxin I contains 8 non-heme iron and 8 acid-labile sulfur atoms per molecule; ferredoxin II contains 4 non-heme iron and 4 acid-labile sulfur atoms per molecule. Ferredoxin I was found only in photosynthetically grown cells whereas ferredoxin II was present in both light- and dark-grown cells. Ferredoxin II from both light- and dark-grown cells has the same molecular weight (14,500) and absorption spectrum and has 4 iron and 4 acid-labile sulfur atoms per molecule. Low temperature electron paramagnetic resonance spectra of oxidized and photoreduced ferredoxins I and II from R. rubrum were recorded. The EPR spectrum of oxidized ferredoxin II exhibited a single resonance line at g = 2.012. Oxidized ferredoxin I, however, exhibited a spectrum that may arise from the superimposition of two resonance lines near g = 2.012. Photoreduced ferredoxin II displayed a rhombic EPR spectrum with a g value of 1.94. Photoreduced ferredoxin I exhibited a similar EPR spectrum at a temperature of 16 K, but when the temperature was lowered to 4.5 K the spectrum of ferredoxin I changed. This temperature-dependent spectrum may result from a weak spin-spin interaction between two iron-sulfur clusters. These results are consistent with the conclusion that R. rubrum ferredoxins I and II are, respectively, 8 iron/8 sulfur and 4 iron/4sulfur proteins.
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