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 stra in 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 pu blished amino acid sequence [Minami, Y., Wakabayashi, S., Wada, K., Ma tsubara, H., Kerscher, L. & Oesterhelt, D. (1985) J. Biochem. (Tokyo) 97, 745-751], while the holoprotein molecular mass was found to be 115 50 +/- 1.0 Da. The reduced [3Fe-4S](0) cluster was also shown by direc t electrochemistry and magnetic circular dichroic spectroscopy to unde rgo a one-proton uptake reaction as first observed for Azotobacter chr oococcum 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 p rotonation step has been determined by a novel thin film electrochemic al 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., Stephen s, P. J., LaMar, G. N., Gorst, C. M. & Burgess, B. K. (1993) J. Biol. Chem. 268, 25928-25939] and indicates that the polypeptide chain aroun d the [3Fe-4S] cluster controls this reaction. Although this appears t o be only the second reported case of protonation at or near the reduc ed [3Fe-4S](0) cluster, its observation in S, acidocaldarius ferredoxi n raises the question of the generality of this chemistry for 3Fe clus ters. The similarity of the pK(a) to the estimated intracellular pH of S. acidocaldarius strongly suggests a physiological role for this pro cess.