Intramolecular Electron Transfer in Trimethylamine Dehydrogenase:  A Thermodynamic Analysis

Abstract

Within the enzyme trimethylamine dehydrogenase [TMADH], intramolecular electron transfer occurs between a fully reduced covalently bound 6-S-cysteinylflavin [FMN] cofactor, and an oxidized iron-sulfur [4Fe-4S]2+ center. When the enzyme is reduced by substrate trimethylamine, the kinetics of this intramolecular electron transfer [ET] reaction are biphasic, suggesting that ET occurs via two alternative processes [Falzon, L., & Davidson, V.L. (1996) Biochemistry 35, 2445-2452]. The formation of the FMN semiquinone was monitored by stopped-flow spectroscopy, and the two rate constants for the biphasic reaction were determined at temperatures ranging from 12 to 37 degrees C. Analysis of these rate constants by ET theory yielded values of 2.2 eV for the reorganizational energy [lambda] associated with each reaction and electronic coupling [H(AB)] of 5.9 and 47 cm-1 for the slower and faster ET reactions, respectively. The analysis also predicted average theoretical distance between the two redox centers of 12.3 A for the slower reaction and 8.1 A for the faster reaction. These predicted distances correlate well with the known crystal structure of TMADH and the most efficient pathways for ET that were predicted from the known structure using the Greenpath program. This analysis suggests that for each reaction the ET event is rate-limiting, but coupled to a highly unfavorable non-ET process, and that binding of a second molecule of substrate to reduced TMADH decreases the efficiency of the intramolecular ET.

Electron Transfer in Nitrogenase Analyzed by Marcus Theory: Evidence for Gating by MgATP

“…Interestingly, while the activation enthalpy (Δ H ⧧ ) for the reaction is similar to that observed for the MgATP-dependent reaction in wild-type nitrogenase, the activation entropy (Δ S ⧧ ) is dramatically different, being of opposite sign (Table ). Negative values of the activation entropy have been observed for other protein−protein electron transfer reactions, although the meaning of these values in not clear ( , ).…”

Evidence for Concerted Electron Proton Transfer in Charge Recombination between FADH– and 306Trp• in Escherichia coli Photolyase

“…Higher reorganization energies of 1.8 and 2.2 eV have been measured for ET with flavin as one of the reactants and were assigned to electrostatics in the active site and to required conformational changes, respectively. 69,70 Calculations and experiments on photoinduced ET between a flavin and tryptophan predict a λ of 0.7À2.1 eV. 71,72 The values of λ larger than 1.3 eV in these studies are mainly due to solvent contributions to the outer sphere reorganization energy and, to a lesser extent, to involvement of the flavin excited state.…”

Redox Cycles in Trimethylamine Dehydrogenase and Mechanism of Substrate Inhibition

“…The spectroscopic and potentiometric data for wild-type TMADH provide a framework for rationalizing the observed steady-state behavior of the enzyme. Previous workers have proposed a second substrate binding site in the enzyme whose occupation leads to substrate inhibition by attenuating internal electron transfer from the flavin to the 4Fe-4S center ( , ). However, the X-ray structure of TMADH in complex with TMAC provides no evidence for a second binding site ( , ), and studies with 14 C TMA reveal that no more than 1 equiv of substrate is bound to the reduced enzyme ().…”

“…Biochemistry, Vol. 38 only a small quantity of anionic flavin semiquinone. Under these conditions the enhanced absorption at 365 nm seen at high TMA concentrations is lost (Figure 7, A).…”