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Titolo:
ELECTRON-TRANSFER FROM FLAVIN TO IRON IN THE PSEUDOMONAS-OLEOVORANS RUBREDOXIN REDUCTASE-RUBREDOXIN ELECTRON-TRANSFER COMPLEX
Autore:
LEE HJ; BASRAN J; SCRUTTON NS;
Indirizzi:
UNIV LEICESTER,DEPT BIOCHEM,UNIV RD LEICESTER LE1 7RH LEICS ENGLAND UNIV LEICESTER,DEPT BIOCHEM LEICESTER LE1 7RH LEICS ENGLAND
Titolo Testata:
Biochemistry (Easton)
fascicolo: 44, volume: 37, anno: 1998,
pagine: 15513 - 15522
SICI:
0006-2960(1998)37:44<15513:EFFTII>2.0.ZU;2-A
Fonte:
ISI
Lingua:
ENG
Soggetto:
ENZYMATIC OMEGA-OXIDATION; METHYLAMINE DEHYDROGENASE; TRIMETHYLAMINE DEHYDROGENASE; CYTOCHROME C-551I; PROTON-TRANSFER; PROTEIN; AMICYANIN; DIIRON; HYDROXYLASE; SUBSTRATE;
Tipo documento:
Article
Natura:
Periodico
Settore Disciplinare:
Science Citation Index Expanded
Citazioni:
38
Recensione:
Indirizzi per estratti:
Citazione:
H.J. Lee et al., "ELECTRON-TRANSFER FROM FLAVIN TO IRON IN THE PSEUDOMONAS-OLEOVORANS RUBREDOXIN REDUCTASE-RUBREDOXIN ELECTRON-TRANSFER COMPLEX", Biochemistry (Easton), 37(44), 1998, pp. 15513-15522

Abstract

Rubredoxin reductase (RR) and rubredoxin form a soluble and physiological eT complex. The complex provides reducing equivalents for a membrane-bound omega-hydroxylase, required for the hydroxylation of alkanesand related compounds, The gene (alkT) encoding RR has been overexpressed and the enzyme purified in amounts suitable for studies of eT by stopped-flow spectroscopy. The eT reactions from NADH to the flavin ofRR and from reduced RR to the 1Fe and 2Fe forms of rubredoxin have been characterized by transient kinetic and thermodynamic analysis. The reductive half-reaction proceeds in a one-step reaction involving oxidized enzyme and a two-electron-reduced enzyme-NAD(+) charge-transfer complex. Flavin reduction is observed at 450 nm and charge-transfer formation at 750 nm; both steps are hyperbolically dependent on NADH concentration. The limiting flavin reduction rate (180 +/- 4 s(-1)) is comparable to the Limiting rate for charge-transfer formation (189 +/- 7 s(-1)) and analysis at 450 and 750 nm yielded enzyme-NADH dissociationconstants of 36 +/- 2 and 43 +/- 5 mu M, respectively. Thermodynamic analysis of the reductive half-reaction yielded values for changes in entropy (Delta S double dagger = -65.8 +/- 2.2 J mol(-1) K-1), enthalpy (Delta H double dagger = 37.8 +/- 0.6 kJ mol(-1)) and Gibbs free energy (Delta G double dagger = 57.5 +/- 0.7 kJ mol(-1) at 298 K) during hydride ion transfer to the flavin N5 atom. Spectral analysis of mixtures of 1Fe or 2Fe rubredoxin and RR suggest that conformational changes accompany eT complex assembly. Both the 1Fe (nonphysiological) and 2Fe (physiological) forms of rubredoxin were found to oxidize two electron-reduced rubredoxin reductase with approximately equal facility. Rates for the reduction of rubredoxin are hyperbolically dependent on rubredoxin concentration and the limiting rates are 72.7 +/- 0.6 and 55.2 +/- 0.3 s(-1) for the 1Fe and 2Fe forms, respectively. Analysis of the temperature dependence of eT to rubredoxin using eT theory revealedthat the reaction is not adequately described as a nonadiabatic eT reaction (H-AB much greater than 80 cm(-1)). eT to both the 1Fe and 2Fe forms of rubredoxin is therefore gated by an adiabatic process that precedes the eT reaction from flavin to iron. Possible origins of this adiabatic event ape discussed.

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Documento generato il 05/12/20 alle ore 20:08:13