Stopped-Flow Kinetic Studies of Flavin Reduction in Human Cytochrome P450 Reductase and Its Component Domains

Abstract

The reduction by NADPH of the FAD and FMN redox centers in human cytochrome P450 reductase and its component domains has been studied by rapid-mixing, stopped-flow spectroscopy. Reduction of the isolated FAD-domain occurs in three kinetically resolvable steps. The first represents the rapid formation (>500 s(-)(1)) of a charge-transfer species between oxidized FAD and NADPH. This is followed by an isomerization ( approximately 200 s(-)(1)) to a second charge-transfer species, characterized by a more intense absorption in the long-wavelength region. The third step represents hydride transfer from NADPH to FAD and is accompanied by a change in the tryptophan fluorescence of the FAD-domain. Flavin reduction is reversible, and the observed rate of hydride transfer displays a complex dependence on NADPH concentration. Two-electron-reduced FAD-domain is active in electron transfer reactions with the isolated FMN domain through the formation of a weakly associating electron transfer complex. Reduction of the CPR by NADPH occurs without direct spectral evidence for the formation of charge-transfer species, although the presence of such species is inferred indirectly. Transfer of the first hydride ion leads to the accumulation of a blue di-semiquinoid species of the reductase, indicating rapid transfer of one electron to the FMN domain. The di-semiquinoid species decays on transfer of the second hydride ion. A third phase is seen following prolonged incubation with NADPH and is assigned to a series of equilibration reactions between different redox species of the enzyme as the system relaxes to its thermodynamically most stable state. As with the isolated FAD-domain, the first hydride transfer in the reductase shows a complex dependence on NADPH concentration. At high NADPH concentration, the observed rate of hydride transfer is slow (approximately 20 s(-1)), and this attenuated rate is attributed to the reversible formation of an less active complex resulting from the binding of a second molecule of NADPH. The kinetic data are discussed with reference to the potentiometric studies on the enzyme and its component domains presented in the preceding paper in this issue [Munro, A., Noble, M., Robledo, L., Daff, S., and Chapman, S. (2001) Biochemistry 40, 1956-1963].

Stopped-flow kinetic studies of electron transfer in the reductase domain of neuronal nitric oxide synthase: re-evaluation of the kinetic mechanism reveals new enzyme intermediates and variation with cytochrome P450 reductase

“…The data indicate that a blue di-semiquinoid form of nNOS reductase domain, containing semiquinone FMN (FMN sq ) and semiqinone FAD (FAD sq ), does not accumulate at significant levels in the stopped-flow experiment, even though it forms under equilibrium conditions during titration experiments with NADPH [24]. This finding is in stark contrast to stopped-flow studies on CPR where, owing to the relatively fast rate of interflavin electron transfer in CPR [25], the di-semiquinoid species forms immediately following hydride transfer from NADPH to FAD. The inability to populate the blue di-semiquinoid form of NOS reductase suggests that there is a kinetic restriction on forming this intermediate.…”

“…In steady-state assays, the enzyme displayed the usual CaMdependent stimulation of cytochrome c reductase activity and determined values of apparent K m and k cat were as published previously [31]. The enzyme was purified predominantly in the ' air-stable ' semiquinone state ; prior to all stopped-flow kinetic studies, nNOS reductase was oxidized with potassium hexacyanoferrate using the procedure reported in our recent work on human CPR [25]. Titration of oxidized enzyme to the twoelectron level with NADPH generates the blue-semiquinone species of nNOS reductase, thus establishing that the enzyme, as purified, has identical properties to those reported by other workers.…”

“…As discussed below, the kinetic restriction that prevents formation of the blue semiquinone in stopped-flow experiments with nNOS reductase is attributed to the slow release of NADP + from reduced enzyme. In human CPR, NADP + release is relatively fast [25], allowing rapid internal electron transfer to form the blue di-semiquinoid intermediate. In the Trp'(' His mutant of CPR, NADP + release is impaired, and the blue di-semiquinoid intermediate does not form in stopped-flow studies, although it does accumulate under equilibrium titration conditions following reduction at the two-electron level with sodium dithionite or NADPH [26].…”

“…Also, like CPR, the NOS reductase domain must be reduced beyond a one-electronreduced state to enable efficient electron transfer to the oxygenase domain [23,24]. Of the mammalian diflavin enzymes, stoppedflow and steady-state kinetic studies of CPR are perhaps the most advanced [25][26][27][28]. Detailed mechanisms for electron flow and the effects of ligand binding and ionic strength have been reported [29,30].…”

“…We show that interflavin electron transfer is relatively slow in nNOS reductase and is probably gated by NADP + -release. The blue di-semiquinoid species (a prominent intermediate in the CPR reaction [25,27]) does not accumulate in stopped-flow studies with NOS, despite its formation in equilibrium titration experiments. Our data reveal that the mechanism of electron transfer in nNOS reductase needs to be re-evaluated.…”