Fate of the (2Fe-2S)²⁺ cluster of Escherichia coli biotin synthase during reaction: A Mössbauer characterization

Biotin synthase, the enzyme which catalyzes the last step of the biosynthesis of biotin, contains only (2Fe-2S)²⁺ clusters when isolated under aerobic conditions. Previous results showed that reduction by dithionite or photoreduced deazaflavin converts the (2Fe-2S)²⁺ to (4Fe-4S)^2+,+. However, until now, no detailed investigation concerning the fate of the (2Fe-2S)²⁺ during reduction under assay conditions (NADPH, flavodoxin, flavodoxin reductase) has been realized. Here, we show by Mössbauer spectroscopy on a partially purified fraction overexpressing the enzyme that, in the presence of a S^2- source and Fe²⁺, there is conversion of the predominant (2Fe-2S)²⁺ clusters into a 1:1 mixture of (2Fe-2S)²⁺ and (4Fe-4S)²⁺. No change in this cluster composition was observed in the presence of the physiological reducing system. When the reaction was allowed to proceed by addition of the substrate dethiobiotin, the (4Fe-4S)²⁺ was untouched whereas the (2Fe-2S)²⁺ was degraded into a new species. This is consistent with the hypothesis that the reduced (4Fe-4S) cluster is involved in mediating the cleavage of AdoMet and that the (2Fe-2S)²⁺ is the sulfur source for biotin.