2Fe-2S] to [4Fe-4S] cluster conversion in Escherichia coli biotin synthase.

Abstract

The type and properties of the Fe-S cluster in recombinant Escherichia coli biotin synthase have been investigated in as-prepared and dithionite-reduced samples using the combination of UV-visible absorption and variable-temperature magnetic circular dichroism (VTMCD), EPR, and resonance Raman spectroscopies. The results confirm the presence of one S = 0 [2Fe-2S]2+ cluster in each subunit of the homodimer in aerobically purified samples, and the Fe-S stretching frequencies suggest incomplete cysteinyl-S coordination. However, absorption and resonance Raman studies show that anaerobic reduction with dithionite in the presence of 60% (v/v) ethylene glycol or glycerol results in near-stoichiometric conversion of two [2Fe-2S]2+ clusters to form one S = 0 [4Fe-4S]2+ cluster with complete cysteinyl-S coordination. The stoichiometry and ability to effect reductive cluster conversion without the addition of iron or sulfide suggest that the [4Fe-4S]2+ cluster is formed at the subunit interface via reductive dimerization of [2Fe-2S]2+ clusters. EPR and VTMCD studies indicate that more than 50% of the Fe is present as [4Fe-4S]+ clusters in samples treated with 60% (v/v) glycerol after prolonged dithionite reduction. The [4Fe-4S]+ cluster exists as a mixed spin system with S = 1/2 (g = 2. 044, 1.944, 1.914) and S = 3/2 (g = 5.6 resonance) ground states. Subunit-bridging [4Fe-4S]2+,+ clusters, that can undergo oxidative degradation to [2Fe-2S]2+ clusters during purification, are proposed to be a common feature of Fe-S enzymes that require S-adenosylmethionine and function by radical mechanisms involving the homolytic cleavage of C-H or C-C bonds, i.e., biotin synthase, anaerobic ribonucleotide reductase, pyruvate formate lyase, lysine 2, 3-aminomutase, and lipoic acid synthase. The most likely role for the [4Fe-4S]2+,+ cluster lies in initiating the radical mechanism by directly or indirectly facilitating reductive one-electron cleavage of S-adenosylmethionine to form methionine and the 5'-deoxyadenosyl radical. It is further suggested that oxidative cluster conversion to [2Fe-2S]2+ clusters may play a physiological role in these radical enzymes, by providing a method of regulating enzyme activity in response to oxidative stress, without irreversible cluster degradation.