TMADH

Like PDR, trimethylamine dehydrogenase (TMADH) from Methylo-philus methylotrophus W3A1 provides an example of a system in which an iron-sulfur center, in this case a [4Fe-4S] cluster, interacts... 

Loechel et al. [20] Trimethylamine Fish Trimethylamine dehydrogenase (TMADH) (Dimethylamine) methylene ferrocene (DMAMFe)... 

TMADH Trimethylamine FejSj ETF TIM FMN Loop-helix EC4S4... 

These results are consistent with the behavior of the redox potential of TMADH At pH 7, the two potentials for FMN, E x/sq andE sq/red... 

The electronic coupling has been calculated for each of the flavin electron transfer complexes described in this chapter and is described below, with the exceptions of CPR, TMADH and PDR. For these three proteins, the two redox cofactors are in direct van der Waals contact, either between the C-7 and C-8 methyl groups of two flavins (CPR), or between the flavin C-8 methyl and a cysteinyl sulfur ligand to the iron-sulfur center (TMADH and PDR). In these cases the coupling between the redox centers should be maximal and the electron transfer rates should depend only on the driving foree and reorganization energy for the electron transfer proeesses. 

The foeus of this chapter is the soluble electron transfer complex formed between the nieotinamide-independent trimethylamine dehydrogenase (TMADH) and eleetron transferring flavoprotein (ETF). Recent studies of this physiological electron transfer complex have provided invaluable insight into (i) the mechanisms of inter and intraprotein electron transfer between flavin and Fe/S centers, (ii) the role of dynamics in interprotein electron transfer and (hi) quantum meehanieal mechanisms for the cleavage of substrate C-H bonds and the subsequent transfer of reducing equivalents to flavin redox centers. Brief mention is made of early structural and cofactor analyses for this redox system, but more detailed accounts of this work can be found in earlier reviews on the subjeet (e.g. Steenkamp and Mathews, 1992). 

Early crystallographic studies of TMADH provided data from two derivatives at 6 resolution that revealed the domain structure and certain elements of secondary structure (Lim et al., 1982 Lim et al., 1984). Higher resolution data at 2.4 resolution have been collected and the structure solved by the multiple isomorphous replacement method with anomolous scattering (Lim et al., 1986). Analysis of the diffraction pattern lead to the identification of ADP as the third cofactor in TMADH. At the time the 2.4 data set was analysed, there was no sequence information available for TMADH (Lim et al., 1986), except for a 12 residue peptide which contained the covalently bound flavin (Kenney et al., 1978). Gas-phase sequencing of isolated peptides initially provided 80% of the primary sequence of... 

TMADH. The chemically determined sequence was found to be approximately 80% identical with the ix-ray deduced sequence (Barber et al., 1992). Aeeounts of some of the earlier crystallographic studies have been reviewed (Steenkamp and Mathews, 1992). The structure of TMADH has now been solved at 2.4 resolution (Brookhaven code 2TMD) and more reeently at 1.7 resolution (Mathews et al., unpublished) and these studies were aided by the full determination of the primary sequence through gene sequeneing methods (Boyd et al., 1992). 

FIGURE 3. Ca trace illustrating the structural similarity between the small domain of TMADH (solid line) and the NADPH-binding domain of glutathione reductase (dashed line). 

Full reduction of TMADH requires three eleetrons per subunitotwo for reduetion of the 6-.S-cysteinyl FMN and a third for reduction of the... 

The route of electron flow through the TMADH-ETF electron transfer complex, FMN —> 4Fe-4S —> ETF, is consistent with the known reduction potentials of the enzyme cofactors and studies in which selective inactivation of the centers in TMADH has been achieved. The reduction... 

FIGURE 4. Spectral forms of intermediates in the reductive half-reaction of TMADH. Panel A, photodiode array analysis of the reaction of TMADH with TMA. Panel B, Denconvoluted spectra for the intermediates of the reductive half-reaction. Spectrum A, oxidised enzymes spectrum B, reduced enzyme (dihydroflavin) spectrum C, reduced enzyme (flavin semi-quinone/reduced iron-sulfur centre) spectrum D, spin-interacting state. 

FIGURE 5. Proposed reaction mechanisms for flavin-catalysed oxidation of amines. A, the carbanion mechanism initially proposed for TMADH (Rohlfs and Hille, 1994). B, the amminium cation radical mechanism, as originally proposed for monoamine oxidase (Silver-man, 1995) although only the pathway passing through a transient covalent intermediate is shown, several alternative pathways for breakdown of the initial flavin semiquinone/... 

Although at pH 8 the electron distribution favours the formation of flavin semiquinone and reduced iron-sulfur center, the magnetic moments of the two redox centers do not interact. At pH 10, however, 2-electron-reduced TMADH exhibits the EPR spectrum diagnostic of the spin-mteracting state. In a more detailed analysis using the pH-jump technique, the interconversion of three states of TMADH [state 1, dihy-droflavin-oxidised 4Fe-4S center (formed at pH 6) state 2, flavin semi-quinone-reduced 4Fe-4S center (formed at pH 8) state 3, spin interacting state (formed at pH 10)] were studied in both H2O and D2O (Rohlfs et al., 1995). The kinetics were found to be consistent with a reaction mechanism that involves sequential protonation/deprotonation and electron transfer events (Figure 6). Normal solvent kinetic isotope effects were observed and proton inventory analysis revealed that at least one proton is involved in the reaction between pH 6 and 8 and at least two protons are involved between pH 8 and 10. At least three protonation/... 

---