The genes encoding the Escherichia coli flavodoxin NADP ϩ oxidoreductase (FLDR) and flavodoxin (FLD) have been overexpressed in E. coli as the major cell proteins (at least 13.5% and 11.4% of total soluble protein, respectively) and the gene products purified to homogeneity. The FLDR reduces potassium ferricyanide with a k cat of 1610.3 min Ϫ1 and a K m of 23.6 µM, and cytochrome c with a k cat of 141.3 min Ϫ1 and a K m of 17.6 µM. The cytochrome c reductase rate is increased sixfold by addition of FLD and an apparent K m of 6.84 µM was measured for the affinity of the two flavoproteins. The molecular masses of FLDR and FLD apoproteins were determined as 27648 Da and 19606 Da and the isolectric points as 4.8 and 3.5, respectively. The mass of the FLDR is precisely that predicted from the atomic structure and indicates that residue 126 is arginine, not glutamine as predicted from the gene sequence. FLDR and FLD were covalently crosslinked using 1-ethyl-3(dimethylamino-propyl) carbodiimide to generate a catalytically active heterodimer. The midpoint reduction potentials of the oxidised/semiquinone and semiquinone/hydroquinone couples of both FLDR (Ϫ308 mV and Ϫ268 mV, respectively) and FLD (Ϫ254 mV and Ϫ433 mV, respectively) were measured using redox potentiometry. This confirms the electron-transfer route as NADPH→FLDR→FLD. Binding of 2′ adenosine monophosphate increases the midpoint reduction potentials for both FLDR couples. These data highlight the strong stabilisation of the flavodoxin semiquinone (absorption coefficient calculated as 4933 M Ϫ1 cm Ϫ1 at 583 nm) with respect to the hydroquinone state and indicate that FLD must act as a single electron shuttle from the semiquinone form in its support of cellular functions, and to facilitate catalytic activity of microsomal cytochromes P-450 heterologously expressed in E. coli. Kinetic studies of electron transfer from FLDR/FLD to the fatty acid oxidase P-450 BM3 support this conclusion, indicating a ping-pong mechanism. This is the first report of the potentiometric analysis of the full E. coli NAD(P)H/FLDR/FLD electron-transfer chain; a complex critical to the function of a large number of E. coli redox systems.Keywords : flavodoxin ; flavodoxin NADP ϩ oxidoreductase; redox potentiometry; enzyme kinetics; cytochrome P-450.The Escherichia coli flavodoxin NADP ϩ oxidoreductase (FLDR or flavodoxin reductase) and flavodoxin (FLD) are the two flavin-containing components of a short electron-transfer chain from NADPH, which provides electrons for the function of the biotin synthase [1] and cobalamin-dependent methionine synthase systems [2]. The enzymes are also required during anaerobic growth of the organism, participating in the pyruvate formate/lyase system of E. coli Ϫ a crucial mechanism for the anaerobic generation of pyruvate for glycolysis [3] and in the generation of deoxyribonucleotides through the enzyme anaerobic ribonucleotide reductase [4]. Recently, the FLDR/FLD system has also been recognised as the E. coli 'reductase', which can support the functio...
We have isolated a soluble cytochrome from Shewanella oneidensis that contains eight covalently attached heme groups and determined its crystal structure. One of these hemes exhibits novel ligation of the iron atom by the epsilon-amino group of a lysine residue, despite its attachment via a typical CXXCH motif. This heme is most likely the active site for tetrathionate reduction, a reaction catalyzed efficiently by this enzyme.
The gene encoding Escherichia coli biotin synthase (bioB) has been expressed as a histidine fusion protein, and the protein was purified in a single step using immobilized metal affinity chromatography. The His 6 -tagged protein was fully functional in in vitro and in vivo biotin production assays. Analysis of all the published bioB sequences identified a number of conserved residues. Single point mutations, to either serine or threonine, were carried out on the four conserved (Cys-53, Cys-57, Cys-60, and Cys-188) and one non-conserved (Cys-288) cysteine residues, and the purified mutant proteins were tested both for ability to reconstitute the [2Fe-2S] clusters of the native (oxidized) dimer and enzymatic activity. The C188S mutant was insoluble. The wild-type and four of the mutant proteins were characterized by UV-visible spectroscopy, metal and sulfide analysis, and both in vitro and in vivo biotin production assays. The molecular masses of all proteins were verified using electrospray mass spectrometry. The results indicate that the His 6 tag and the C288T mutation have no effect on the activity of biotin synthase when compared with the wild-type protein. The C53S, C57S, and C60S mutant proteins, both as prepared and reconstituted, were unable to covert dethiobiotin to biotin in vitro and in vivo. We conclude that three of the conserved cysteine residues (Cys-53, Cys-57, and Cys-60), all of which lie in the highly conserved "cysteine box" motif, are crucial for [Fe-S] cluster binding, whereas Cys-188 plays a hitherto unknown structural role in biotin synthase.
The structure of the Escherichia coli flavodoxin NADP + oxidoreductase (FLDR) places three arginines (R144, R174 and R184) in the proposed NADPH-binding site. Mutant enzymes produced by site-directed mutagenesis, in which each arginine was replaced by neutral alanine, were characterized. All mutants exhibited decreased NADPH-dependent cytochrome c reductase activity (R144A, 241.6 min V " ; R174A, 132.1 min V " ; R184A, 305.5 min V " versus wild type, 338.9 min V ") and increased K m for NADPH (R144A, 5.3 µM ; R174A, 20.2 µM ; R184A, 54.4 µM versus wild type, 3.9 µM). The k cat value for NADH-dependent cytochrome c reduction was increased for R174A (42.3 min V ") and R184A (50.4 min V ") compared with the wild type (33.0 min V "), consistent with roles for R174 and R184 in discriminating between NADPH\NADH by interaction with the adenosine ribose 2h-phosphate. Stopped-flow studies indicated that affinity (K d ) for NADPH was markedly reduced in mutants R144A (635 µM) and R184A (2.3 mM) compared with the wild
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