IDENTIFICATION OF THE IRON-SULFUR CLUSTERS IN A FERREDOXIN FROM THE ARCHAEON SULFOLOBUS-ACIDOCALDARIUS - EVIDENCE FOR A REDUCED [3FE-4S] CLUSTER WITH PH-DEPENDENT ELECTRONIC-PROPERTIES

A ferredoxin isolated from the archaeon Sulfolobus acidocaldarius strain DSM 639 has been shown to contain one [3Fe-4S](1+/0) cluster with a reduction potential of -275 mV and one [4Fe-4S](2+/1+) cluster with a reduction potential of -529 mV at pH 6.4, in the temperature range 0-50 degrees C. The monomer molecular mass was confirmed to be 10907.5 +/- 1.0 Da by electrospray mass spectrometry, as calculated from the published amino acid sequence [Minami, Y., Wakabayashi, S., Wada, K., Matsubara, H., Kerscher, L. & Oesterhelt, D. (1985) J. Biochem. (Tokyo) 97, 745-751], while the holoprotein molecular mass was found to be 11550 +/- 1.0 Da. The reduced [3Fe-4S](0) cluster was also shown by direct electrochemistry and magnetic circular dichroic spectroscopy to undergo a one-proton uptake reaction as first observed for Azotobacter chroococcum ferredoxin I [George, S. J., Richards, A. J. M., Thomson, A. J. & Yates, M. G. (1984) Biochem. J. 224, 247-251]. The pK(a) of the protonation step has been determined by a novel thin film electrochemical method to be 5.8. This is significantly different from the pK(a) of 7.7 determined for A. vinelandii ferredoxin I [Shen, B., Martin, L. L.: Butt, J. N., Armstrong, F. A., Stout, C. D., Jensen, J. M., Stephens, P. J., LaMar, G. N., Gorst, C. M. & Burgess, B. K. (1993) J. Biol. Chem. 268, 25928-25939] and indicates that the polypeptide chain around the [3Fe-4S] cluster controls this reaction. Although this appears to be only the second reported case of protonation at or near the reduced [3Fe-4S](0) cluster, its observation in S, acidocaldarius ferredoxin raises the question of the generality of this chemistry for 3Fe clusters. The similarity of the pK(a) to the estimated intracellular pH of S. acidocaldarius strongly suggests a physiological role for this process.