Document Detail


Characterisation of flavodoxin NADP+ oxidoreductase and flavodoxin; key components of electron transfer in Escherichia coli.
MedLine Citation:
PMID:  9839946     Owner:  NLM     Status:  MEDLINE    
Abstract/OtherAbstract:
The genes encoding the Escherichia coli flavodoxin NADP+ 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 kcat of 1610.3 min(-1) and a Km of 23.6 microM, and cytochrome c with a kcat of 141.3 min(-1) and a Km of 17.6 microM. The cytochrome c reductase rate is increased sixfold by addition of FLD and an apparent Km of 6.84 microM 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 isoelectric 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 (-308 mV and -268 mV, respectively) and FLD (-254 mV and -433 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(-1) cm(-1) 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.
Authors:
L McIver; C Leadbeater; D J Campopiano; R L Baxter; S N Daff; S K Chapman; A W Munro
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Publication Detail:
Type:  Journal Article; Research Support, Non-U.S. Gov't    
Journal Detail:
Title:  European journal of biochemistry / FEBS     Volume:  257     ISSN:  0014-2956     ISO Abbreviation:  Eur. J. Biochem.     Publication Date:  1998 Nov 
Date Detail:
Created Date:  1998-12-22     Completed Date:  1998-12-22     Revised Date:  2007-07-23    
Medline Journal Info:
Nlm Unique ID:  0107600     Medline TA:  Eur J Biochem     Country:  GERMANY    
Other Details:
Languages:  eng     Pagination:  577-85     Citation Subset:  IM    
Affiliation:
Department of Chemistry, The University of Edinburgh, UK.
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MeSH Terms
Descriptor/Qualifier:
Base Sequence
Cross-Linking Reagents / chemistry
Cytochrome P-450 Enzyme System / metabolism
DNA Primers
Electron Transport
Escherichia coli / enzymology,  metabolism*
Flavodoxin / chemistry,  metabolism*
NADH, NADPH Oxidoreductases / chemistry,  metabolism*
Oxidation-Reduction
Potentiometry
Chemical
Reg. No./Substance:
0/Cross-Linking Reagents; 0/DNA Primers; 0/Flavodoxin; 9035-51-2/Cytochrome P-450 Enzyme System; EC 1.6.-/NADH, NADPH Oxidoreductases; EC 1.6.-/flavodoxin NADPH oxidoreductase

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