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 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.
Tse Sum Bui
Bernadette
B
Laboratoire de Chimie Organique Biologique, Université Paris VI, UMR CNRS 7613, 4 Place Jussieu, 75252 Paris Cedex 05, France.
Benda
Rüdiger
R
Schünemann
Volker
V
Florentin
Dominique
D
Trautwein
Alfred X
AX
Marquet
Andrée
A
D000645:Ammonium Sulfate / Q000494:pharmacology; D004305:Dose-Response Relationship, Drug; D004926:Escherichia coli / Q000201:enzymology*; D005182:Flavin-Adenine Dinucleotide / Q000494:pharmacology; D007501:Iron / Q000378:metabolism; D008956:Models, Chemical; D009249:NADP / Q000378:metabolism; D015204:Spectroscopy, Mossbauer / Q000379:methods*; D013347:Subcellular Fractions; D013455:Sulfur / Q000378:metabolism; D013466:Sulfurtransferases / Q000737:chemistry*
D005182:Flavin-Adenine Dinucleotide; D009249:NADP; D013455:Sulfur; D007501:Iron; D013466:Sulfurtransferases; C057985:biotin synthetase; D000645:Ammonium Sulfate