ELECTRON-TRANSFER FROM FLAVIN TO IRON IN THE PSEUDOMONAS-OLEOVORANS RUBREDOXIN REDUCTASE-RUBREDOXIN ELECTRON-TRANSFER COMPLEX

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.