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-25) 2 + clusters when isolated under aerobic conditions. Previous results showed that reduction by dithionite or photoreduced deazaflavin converts the (2Fe-25 ) 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 Mossbauer spectroscopy on a partially purified fraction overexpressing the enzyme that, in the presence of a S 2 - source and Fe 2 + , 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.