Iron−Sulfur Clusters of Biotin Synthase In Vivo:  A Mössbauer Study

Abstract

Biotin synthase, the enzyme that catalyzes the last step of the biosynthesis of biotin, contains only [2Fe-2S](2+) clusters when isolated under aerobic conditions. Previous results showed that reconstitution with an excess of FeCl(3) and Na(2)S under reducing and anaerobic conditions leads to either [4Fe-4S](2+), [4Fe-4S](+), or a mixture of [4Fe-4S](2+) and [2Fe-2S](2+) clusters. To determine whether any of these possibilities or other different cluster configuration could correspond to the physiological in vivo state, we have used (57)Fe Mössbauer spectroscopy to investigate the clusters of biotin synthase in whole cells. The results show that, in aerobically grown cells, biotin synthase contains a mixture of [4Fe-4S](2+) and [2Fe-2S](2+) clusters. A mixed [4Fe-4S](2+):[2Fe-2S](2+) cluster form has already been observed under certain in vitro conditions, and it has been proposed that both clusters might each play a significant role in the mechanism of biotin synthase. Their presence in vivo is now another argument in favor of this mixed cluster form.

Radical S-Adenosylmethionine Enzyme Coproporphyrinogen III Oxidase HemN

“…Furthermore, all AdoMet radical proteins require S-adenosylmethionine as a cofactor to initiate radical-based catalysis. The iron-sulfur clusters of several members of the family have been characterized by electron paramagnetic resonance (EPR), resonance Raman, and Möss-bauer spectroscopy (7)(8)(9)(10)(11)(12)(13)(14)(15)(16). For pyruvate formate-lyase-activating enzyme (PFL-AE) and biotin synthase (BioB), it was shown using Mössbauer spectroscopy that the [4Fe-4S] cluster contains one iron atom, which is not ligated by a cysteine residue, and that AdoMet binds to this special iron site of the cluster (17,18).…”

Ferric ion (hydr)oxo clusters in the “Venus flytrap” cleft of FbpA: Mössbauer, calorimetric and mass spectrometric studies

“…57 FeCl 3 solution was prepared using a modified literature procedure [35]. 0.0146 g 57 Fe (99.8% purity) was dissolved in 1 mL 37% HCl (over 2 d) and the solution was exposed in air and light until the color turned to brown-yellow.…”

“…and used for cloning and expression [35]. Overexpression and purification of nFbpA (Neisseria Gonorrhoeae) was performed by a literature method [37].…”

“…The mutant genes were completely characterized by DNA sequencing prior to expression in Escherichia coli TOP 10 cells. The expression and purification were carried out using literature procedures [35]. The molar extinction coefficient ε 280 of the pure protein was calculated as 50,390 M -1 cm -1 by addition of 1490 M -1 cm -1 for the inclusion of a tyrosine to the experimental absorption coefficient (ε 280 = 48,900 M -1 cm -1 ) of native holo-nFbpA [38,39].…”

Loss of iron–sulfur clusters from biotin synthase as a result of catalysis promotes unfolding and degradation

“…In addition, the [4Fe-4S] 2+ cluster is rapidly oxidized in the absence of the [2Fe-2S] 2+ cluster and bound substrates [17], most likely resulting in the formation of an inactive apoenzyme following turnover in aerobic bacteria. Mössbauer spectra of whole cell preparations of E. coli expressing recombinant BioB indicate that <35% of the enzyme contains a [4Fe-4S] 2+ cluster, probably due to the air-sensitivity of this cluster [18,19]. Since the biotin requirements of bacteria are very low, on the order of 100-1000 molecules per cell [20], it has been suggested that BioB may be a stoichiometric reactant that is degraded after a single turnover.…”