J. Biol. Chem., Vol. 269, Issue 11, 8001-8006, Mar, 1994
Interaction of NADPH-adrenoferredoxin reductase with NADP+ and adrenoferredoxin. Equilibrium and dynamic properties investigated by proton nuclear magnetic resonance
S Miura and Y Ichikawa
Department of Biochemistry, Kagawa Medical School, Japan.
NADPH-adrenoferredoxin reductase, a flavoprotein from bovine adrenocortical
mitochondria, has been investigated to elucidate the equilibrium and
dynamic properties of the interaction with NADP+ and adrenoferredoxin
(adrenodoxin) using proton NMR spectroscopy. The line width of the signals
from NADP+ depends on the presence of the reductase. The off rate constant
of NADP+ from the reductase is estimated to be about 15-20 s-1 on the basis
of line width measurements. No appreciable difference in off rate is
detected between adenine and nicotinamide moieties of NADP+. Transferred
nuclear Overhauser effect experiments for NADP+ indicate the time-dependent
magnetization transfer profiles with a long lag phase. The proton NMR
spectra during the titration of the reductase with adrenodoxin reveal that
the reductase possesses distinct binding sites for both NADP+ and
adrenodoxin. The sharp resonances in the aromatic region due to His-10 and
His-62 of adrenodoxin were utilized as a probe to explore the interaction
with the reductase. IN the mixture of adrenodoxin and the reductase at the
mol ratio of 6:1, T1 values of the histidine residue in adrenodoxin were
measured by the inversion recovery method. At low ionic strength, T1 values
of the resonances are not affected in the presence or absence of the
reductase. In the presence of the reductase, T1 values of resonances
resulting from the histidine residues become shorter as the concentration
of KCl increases because of rapid exchange between bound and free states.
At low ionic strength (10 mM phosphate buffer), the off rate from the
reductase is estimated to be less than about 4 s-1. The off rate of
adrenodoxin from the reductase could be the rate-limiting step in
cytochrome c reductase activity at low ionic strength.
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