Hj. 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
Rubredoxin reductase (RR) and rubredoxin form a soluble and physiologi
cal eT complex. The complex provides reducing equivalents for a membra
ne-bound omega-hydroxylase, required for the hydroxylation of alkanes
and related compounds, The gene (alkT) encoding RR has been overexpres
sed and the enzyme purified in amounts suitable for studies of eT by s
topped-flow spectroscopy. The eT reactions from NADH to the flavin of
RR and from reduced RR to the 1Fe and 2Fe forms of rubredoxin have bee
n characterized by transient kinetic and thermodynamic analysis. The r
eductive half-reaction proceeds in a one-step reaction involving oxidi
zed enzyme and a two-electron-reduced enzyme-NAD(+) charge-transfer co
mplex. Flavin reduction is observed at 450 nm and charge-transfer form
ation at 750 nm; both steps are hyperbolically dependent on NADH conce
ntration. The limiting flavin reduction rate (180 +/- 4 s(-1)) is comp
arable to the Limiting rate for charge-transfer formation (189 +/- 7 s
(-1)) and analysis at 450 and 750 nm yielded enzyme-NADH dissociation
constants of 36 +/- 2 and 43 +/- 5 mu M, respectively. Thermodynamic a
nalysis of the reductive half-reaction yielded values for changes in e
ntropy (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 ener
gy (Delta G double dagger = 57.5 +/- 0.7 kJ mol(-1) at 298 K) during h
ydride ion transfer to the flavin N5 atom. Spectral analysis of mixtur
es of 1Fe or 2Fe rubredoxin and RR suggest that conformational changes
accompany eT complex assembly. Both the 1Fe (nonphysiological) and 2F
e (physiological) forms of rubredoxin were found to oxidize two electr
on-reduced rubredoxin reductase with approximately equal facility. Rat
es for the reduction of rubredoxin are hyperbolically dependent on rub
redoxin 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 th
e temperature dependence of eT to rubredoxin using eT theory revealed
that the reaction is not adequately described as a nonadiabatic eT rea
ction (H-AB much greater than 80 cm(-1)). eT to both the 1Fe and 2Fe f
orms of rubredoxin is therefore gated by an adiabatic process that pre
cedes the eT reaction from flavin to iron. Possible origins of this ad
iabatic event ape discussed.