Biotin synthase contains two distinct iron-sulfur cluster binding sites: Chemical and spectroelectrochemical analysis of iron-sulfur cluster interconversions

被引:125
作者
Ugulava, NB
Gibney, BR
Jarrett, JT
机构
[1] Univ Penn, Sch Med, Dept Biochem & Biophys, Philadelphia, PA 19104 USA
[2] Univ Penn, Sch Med, Johnson Res Fdn, Philadelphia, PA 19104 USA
关键词
D O I
10.1021/bi0104625
中图分类号
Q5 [生物化学]; Q7 [分子生物学];
学科分类号
071010 ; 081704 ;
摘要
Biotin synthase is an iron-sulfur protein that utilizes AdoMet to catalyze the presumed radical-mediated insertion of a sulfur atom between the saturated C6 and C9 carbons of dethiobiotin. Biotin synthase (BioB) is aerobically purified as a dimer that contains [2Fe-2S](2+) clusters and is inactive in the absence of additional iron and reductants, and anaerobic reduction of BioB with sodium dithionite results in conversion to enzyme containing [4Fe-4S](2+) and/or [4Fe-4S](+) clusters. To establish the predominant cluster forms present in biotin synthase in anaerobic assays, and by inference in Escherichia coli, we have accurately determined the extinction coefficient and cluster content of the enzyme under oxidized and reduced conditions and have examined the equilibrium reduction potentials at which cluster reductions and conversions occur as monitored by UV/visible and EPR spectroscopy. In contrast to previous reports, we find that aerobically purified BioB contains ca. 1.2-1.5 [2Fe-2S](2+) clusters per monomer with epsilon (452) = 8400 M-1 cm(-1) per monomer. Upon reduction, the [2Fe-2S](2+) clusters are converted to [4Fe-4S] clusters with two widely separate reduction potentials of -140 and -430 mV, BioB reconstituted with excess iron and sulfide in 60% ethylene glycol was found to contain two [4Fe-4S](2+) clusters per monomer with epsilon (400) = 30 000 M-1 cm(-1) per monomer and is reduced with lower midpoint potentials of -440 and -505 mV, respectively. Finally, as predicted by the measured redox potentials, enzyme incubated under typical anaerobic assay conditions is repurified containing one [2Fe-2S](2+) cluster and one [4Fe-4S](2+) cluster per monomer. These results indicate that the dominant stable cluster state for biotin synthase is a dimer containing two [2Fe-2S](2+) and two [4Fe-4S](2+) clusters.
引用
收藏
页码:8343 / 8351
页数:9
相关论文
共 46 条
  • [1] IscU as a scaffold for iron-sulfur cluster biosynthesis: Sequential assembly of [2Fe-2S] and [4Fe-4S] clusters in IscU
    Agar, JN
    Krebs, C
    Frazzon, J
    Huynh, BH
    Dean, DR
    Johnson, MK
    [J]. BIOCHEMISTRY, 2000, 39 (27) : 7856 - 7862
  • [2] MECHANISM OF REDUCTIVE ACTIVATION OF COBALAMIN-DEPENDENT METHIONINE SYNTHASE - AN ELECTRON-PARAMAGNETIC RESONANCE SPECTROELECTROCHEMICAL STUDY
    BANERJEE, RV
    HARDER, SR
    RAGSDALE, SW
    MATTHEWS, RG
    [J]. BIOCHEMISTRY, 1990, 29 (05) : 1129 - 1135
  • [3] BARANIAK J, 1989, J BIOL CHEM, V264, P1357
  • [5] BIOTIN SYNTHASE FROM ESCHERICHIA-COLI, AN INVESTIGATION OF THE LOW-MOLECULAR-WEIGHT AND PROTEIN-COMPONENTS REQUIRED FOR ACTIVITY IN-VITRO
    BIRCH, OM
    FUHRMANN, M
    SHAW, NM
    [J]. JOURNAL OF BIOLOGICAL CHEMISTRY, 1995, 270 (32) : 19158 - 19165
  • [6] Pyruvate formate-lyase-activating enzyme:: Strictly anaerobic isolation yields active enzyme containing a [3Fe-4S]+ cluster
    Broderick, JB
    Henshaw, TF
    Cheek, J
    Wojtuszewski, K
    Smith, SR
    Trojan, MR
    McGhan, RM
    Kopf, A
    Kibbey, M
    Broderick, WE
    [J]. BIOCHEMICAL AND BIOPHYSICAL RESEARCH COMMUNICATIONS, 2000, 269 (02) : 451 - 456
  • [7] Biotin synthase mechanism:: on the origin of sulphur
    Bui, BTS
    Florentin, D
    Fournier, F
    Ploux, O
    Méjean, A
    Marquet, A
    [J]. FEBS LETTERS, 1998, 440 (1-2) : 226 - 230
  • [8] Mossbauer studies of Escherichia coli biotin synthase:: evidence for reversible interconversion between [2Fe-2S]2+ and [4Fe-4S]2+ clusters
    Bui, BTS
    Florentin, D
    Marquet, A
    Benda, R
    Trautwein, AX
    [J]. FEBS LETTERS, 1999, 459 (03) : 411 - 414
  • [9] Bui BTS, 2000, EUR J BIOCHEM, V267, P2688
  • [10] DELETION AND COMPLEMENTATION ANALYSIS OF BIOTIN GENE CLUSTER OF ESCHERICHIA-COLI
    CLEARY, PP
    CAMPBELL, A
    [J]. JOURNAL OF BACTERIOLOGY, 1972, 112 (02) : 830 - &