X-ray Scattering Studies of Methylophilus methylotrophus (sp. W3A1) Electron-transferring Flavoprotein

Abstract

Small angle x-ray solution scattering has been used to generate a low resolution, model-independent molecular envelope structure for electron-transferring flavoprotein (ETF) from Methylophilus methylotrophus (sp. W 3 A 1 ). Analysis of both the oxidized and 1-electron-reduced (anionic flavin semiquinone) forms of the protein revealed that the solution structures of the protein are similar in both oxidation states. Comparison of the molecular envelope of ETF from the x-ray scattering data with previously determined structural models of the protein suggests that ETF samples a range of conformations in solution. These conformations correspond to a rotation of domain II with respect to domains I and III about two flexible "hinge" sequences that are unique to M. methylotrophus ETF. The x-ray scattering data are consistent with previous models concerning the interaction of M. methylotrophus ETF with its physiological redox partner, trimethylamine dehydrogenase. Our data reveal that an "induced fit" mechanism accounts for the assembly of the trimethylamine dehydrogenase-ETF electron transfer complex, consistent with spectroscopic and modeling studies of the assembly process. Electron-transferring flavoproteins (ETFs)1 act as physiological electron carriers between degradative enzymes in bacteria and mitochondria and their respective membrane-bound electron transport pathways (1). ETFs are heterodimeric, and they all possess one equivalent of non-covalently bound flavin-adenine dinucleotide (FAD) per ETF dimer, with the exception of ETF from Megasphaera elsdenii, which contains 2 equivalents of FAD per dimer (2). The ETFs from pig, human, Paracoccus denitrificans, and Methylophilus methylotrophus also contain 1 equivalent of AMP per protein dimer (3-6). ETF from M. methylotrophus (sp. W 3 A 1 ) is a highly specific electron carrier that accepts electrons from only one enzyme, trimethylamine dehydrogenase (TMADH) (7). In methylotrophic bacteria, TMADH catalyzes the oxidative demethylation of trimethylamine to dimethylamine and formaldehyde (8). The enzyme enables these organisms to subsist on trimethylamine as their sole source of carbon. M. methylotrophus ETF consists of two subunits with molecular masses of 34 and 29 kDa (9), and it shares considerable sequence identity with bacterial and mammalian ETFs (3, 4). Preliminary crystallographic studies of M. methylotrophus ETF have been reported, but to date no crystallographic structure for the protein is available (10).Mammalian and bacterial ETFs are thought to act as 1-electron carriers, cycling between the oxidized and anionic flavin semiquinone forms. The ETF from M. elsdenii is unusual in that it serves as a 2-electron carrier. ETFs from mammalian sources and P. denitrificans can be reduced (albeit over a long time period) to the dihydroquinone form by reduction with dithionite or during photochemical reduction. M. methylotrophus ETF is readily converted to the semiquinone form in reactions with TMADH or during artificial reduction with dithionite. Further reduction ...

Electron Transfer and Conformational Change in Complexes of Trimethylamine Dehydrogenase and Electron Transferring Flavoprotein

“…The sample-to-detector distance of 1.5 m and protein concentrations of approximately 15 mg/ml were chosen to reveal the scattering behavior in the scattering range 0.01 Å Ϫ1 Յ s Յ 0.08 Å Ϫ1 (s is the modulus of the momentum transfer and defined as s ϭ 2 sin ⌰/, where 2⌰ is the scattering angle and ϭ 1.54 Å is the x-ray wavelength). As established by our previous studies (26,27), this scattering interval is ideal in directly highlighting any differences in the scattering characteristics of the two protein states as a result of possible domain reorganizations. Further information with regards to data collection, reduction, and analysis have been described previously in detail (26,27).…”

“…As established by our previous studies (26,27), this scattering interval is ideal in directly highlighting any differences in the scattering characteristics of the two protein states as a result of possible domain reorganizations. Further information with regards to data collection, reduction, and analysis have been described previously in detail (26,27).…”

“…However, we show in this paper that the kinetics of complex formation cannot be measured using the stopped-flow method owing to the fast time scale of the spectral change (Table I). Formation of the ETF sq ⅐TMADH ox complex can likewise be observed by difference spectroscopy (26), but in this case the absorption changes occur on a time scale that enables characterization by stopped-flow spectroscopy (Table I). Herein, we have extended our work to the Y442F, Y442L, and Y442G TMADH⅐ETF complexes.…”

“…Based on the crystal structure of human ETF (24), our modeling studies (25) suggested that the structure of M. methylotrophus ETF comprises three domains, arranged in a 'Y'-shape, with domains I and III forming a shallow bowl (one rigid body) in which domain II (a second rigid body) sits. We have demonstrated using small-angle x-ray scattering methods that domain II is conformationally mobile and that this flexibility produces an ensemble of ETF structures, only a subset of which can dock onto TMADH (26). Recent studies with human ETF have established domain flexibility as a general feature of the ETF family (27), and these dynamic aspects of ETF structure are likely important in optimizing electron transfer rates from redox donor proteins (26,27).…”

“…We have demonstrated using small-angle x-ray scattering methods that domain II is conformationally mobile and that this flexibility produces an ensemble of ETF structures, only a subset of which can dock onto TMADH (26). Recent studies with human ETF have established domain flexibility as a general feature of the ETF family (27), and these dynamic aspects of ETF structure are likely important in optimizing electron transfer rates from redox donor proteins (26,27).…”