[2FE-2S] TO [4FE-4S] CLUSTER CONVERSION IN ESCHERICHIA-COLI BIOTIN SYNTHASE

The type and properties of the Fe-S cluster in recombinant Escherichia coli biotin synthase have been investigated in as-prepared and dithio nite-reduced samples using the combination of UV-visible absorption an d variable-temperature magnetic circular dichroism (VTMCD), EPR, and r esonance Raman spectroscopies. The results confirm the presence of one S = 0 [2Fe-2S](2+) cluster in each subunit of the homodimer in aerobi cally purified samples, and the Fe-S stretching frequencies suggest in complete cysteinyl-S coordination. However, absorption and resonance R aman studies show that anaerobic reduction with dithionite in the pres ence of 60% (v/v) ethylene glycol or glycerol results in near-stoichio metric conversion of two [2Fe-2S](2+) clusters to form one S = 0 [4Fe- 4S](2+) cluster with complete cysteinyl-S coordination. The stoichiome try and ability to effect reductive cluster conversion without the add ition of iron or sulfide suggest that the [4Fe-4S](2+) cluster is form ed 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-4 S](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 radi cal mechanisms involving the homolytic cleavage of C-H or C-C bonds, i .e., biotin synthase, anaerobic ribonucleotide reductase, pyruvate for mate lyase, lysine 2,3-aminomutase, and lipoic acid synthase. The most likely role for the [4Fe-4S](2+,+) cluster lies in initiating the rad ical mechanism by directly or indirectly facilitating reductive one-el ectron 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 t hese radical enzymes, by providing a method of regulating enzyme activ ity in response to oxidative stress, without irreversible cluster degr adation.