The Oxygen Sensitivity of Spinach Ferredoxin and Other Iron-Sulfur Proteins

Abstract

In the presence of urea and molecular oxygen, spinach ferredoxin is rapidly denatured. The product of this reaction contains substantial amounts of sulfur in the oxidation state of zero covalently bound to the protein. The origin of the sulfur-zero was shown to be the labile sulfide of the native protein. The S(0) could be released from the protein as S2- with a strong reducing agent such as dithiothreitol or as S(0) with cyanide or sulfite anions. Sedimentation analysis showed the protein to be polymeric, existing as a dimer. Ferricyanide could replace oxygen as a source of oxidizing equivalents. With this oxidant the stoichiometry of the reaction was found to be 9 moles per mole of ferredoxin. This has been explained by the oxidation of 5 RSH to 5/2 RSSR and 2 S2- to 2 S(0). It is proposed that the S(0) is bound to the protein as a trisulfide (RSSSR). The ferricyanide titer of a number of iron-sulfur proteins has been determined and correlated with the S(0) content of the urea-ferricyanide denatured proteins. The oxidation stoichiometries are consistent with a generalization of the above formulation. Thus 8 to 10 moles of ferricyanide are consumed by the two-iron proteins and approximately 30 by the bacterial ferredoxins. With the two-iron proteins, 60 to 80% of the labile sulfide was found as bound S(0) while with bacterial ferredoxins about 50% of the labile sulfide was detected as bound S(0). In the case of putidaredoxin it was found that the same amount of S(0) was bound when the protein was air-denatured in the absence of urea and mercaptoethanol as when the protein was denatured in aerobic urea solution. It is proposed that the oxygen sensitivity of many non-heme iron labile-sulfide proteins finds explanation in the oxidation of their mercaptide-sulfide system.