Abstract
The kinetics of electron transfer between trimethylamine dehydrogenase (TMADH) and its physiological acceptor, electron transferring flavoprotein (ETF), has been studied by static and stopped-flow absorbance measurements. The results demonstrate that reducing equivalents are transferred from TMADH to ETF solely through the 4Fe/4S center of the former. The intrinsic limiting rate constant (klim) and dissociation constant (Kd) for electron transfer from the reduced 4Fe/4S center of TMADH to ETF are about 172 s-1 and 10 microM, respectively. The reoxidation of fully reduced TMADH with an excess of ETF is markedly biphasic, indicating that partial oxidation of the iron-sulfur center in 1-electron reduced enzyme significantly reduces the rate of electron transfer out of the enzyme in these forms. The interaction of the two unpaired electron spins of flavin semiquinone and reduced 4Fe/4S center in 2-electron reduced TMADH, on the other hand, does not significantly slow down the electron transfer from the 4Fe/4S center to ETF. From a comparison of the limiting rate constants for the oxidative and reductive half-reactions, we conclude that electron transfer from TMADH to ETF is not rate-limiting during steady-state turnover. The overall kinetics of the oxidative half-reaction are not significantly affected by high salt concentrations, indicating that electrostatic forces are not involved in the formation and decay of reduced TMADH-oxidized ETF complex.
Highlights
Dant, an electron-transferring flavoprotein (ETF), which becomes reduced to the level of the semiquinone (4, 6 – 8)
Previous freeze-quench studies have demonstrated that when TMADH2eq* is mixed with ETFox, the EPR signal arising from the spin-interacting state is lost within a few milliseconds with no concomitant appearance of that for the reduced ironsulfur center, as would be expected if electrons are transferred from the flavosemiquinone of TMADH2eq* to ETFox to give enzyme possessing oxidized flavin and reduced 4Fe/4S center [11]
The present results indicate that when trimethylamine dehydrogenase (TMADH) is treated with phenylhydrazine, rendering the FMN redox-inert [13], the iron-sulfur center can be reduced by dithionite and reoxidized by ETF
Summary
Dant, an electron-transferring flavoprotein (ETF), which becomes reduced to the level of the (anionic) semiquinone (4, 6 – 8). Some TMADH2eq possesses flavin semiquinone and reduced iron-sulfur center at pH 7.0, the magnetic moments of the unpaired spins do not interact as is the case when TMADH is reduced by excess substrate, or by sodium dithionite at high pH or in the presence of the inhibitor tetramethylammonium chloride (6, 9 –11). This spin-interacting form exhibits a unique EPR signal that includes half-field features and is not the sum of the signals for flavin semiquinone and reduced iron-sulfur center; we designate this spin-interacting form as TMADH2eq*. The results are incorporated into a comprehensive kinetic mechanism for the reaction of TMADH
Sign-in/Register to view highlights & summary Talk to us
Join us for a 30 min session where you can share your feedback and ask us any queries you have
Schedule a callDisclaimer: All third-party content on this website/platform is and will remain the property of their respective owners and is provided on "as is" basis without any warranties, express or implied. Use of third-party content does not indicate any affiliation, sponsorship with or endorsement by them. Any references to third-party content is to identify the corresponding services and shall be considered fair use under The CopyrightLaw.
