Contrasting roles for two conserved arginines: Stabilizing flavin semiquinone or quaternary structure, in bifurcating electron transfer flavoproteins
2022; Elsevier BV; Volume: 298; Issue: 4 Linguagem: Inglês
10.1016/j.jbc.2022.101733
ISSN1083-351X
AutoresNishya Mohamed‐Raseek, Anne‐Frances Miller,
Tópico(s)Advanced Fluorescence Microscopy Techniques
ResumoBifurcating electron transfer flavoproteins (Bf ETFs) are important redox enzymes that contain two flavin adenine dinucleotide (FAD) cofactors, with contrasting reactivities and complementary roles in electron bifurcation. However, for both the "electron transfer" (ET) and the "bifurcating" (Bf) FADs, the only charged amino acid within 5 Å of the flavin is a conserved arginine (Arg) residue. To understand how the two sites produce different reactivities utilizing the same residue, we investigated the consequences of replacing each of the Arg residues with lysine, glutamine, histidine, or alanine. We show that absence of a positive charge in the ET site diminishes accumulation of the anionic semiquinone (ASQ) that enables the ET flavin to act as a single electron carrier, due to depression of the oxidized versus. ASQ reduction midpoint potential, E°OX/ASQ. Perturbation of the ET site also affected the remote Bf site, whereas abrogation of Bf FAD binding accelerated chemical modification of the ET flavin. In the Bf site, removal of the positive charge impaired binding of FAD or AMP, resulting in unstable protein. Based on pH dependence, we propose that the Bf site Arg interacts with the phosphate(s) of Bf FAD or AMP, bridging the domain interface via a conserved peptide loop ("zipper") and favoring nucleotide binding. We further propose a model that rationalizes conservation of the Bf site Arg even in non-Bf ETFs, as well as AMP's stabilizing role in the latter, and provides a mechanism for coupling Bf flavin redox changes to domain-scale motion. Bifurcating electron transfer flavoproteins (Bf ETFs) are important redox enzymes that contain two flavin adenine dinucleotide (FAD) cofactors, with contrasting reactivities and complementary roles in electron bifurcation. However, for both the "electron transfer" (ET) and the "bifurcating" (Bf) FADs, the only charged amino acid within 5 Å of the flavin is a conserved arginine (Arg) residue. To understand how the two sites produce different reactivities utilizing the same residue, we investigated the consequences of replacing each of the Arg residues with lysine, glutamine, histidine, or alanine. We show that absence of a positive charge in the ET site diminishes accumulation of the anionic semiquinone (ASQ) that enables the ET flavin to act as a single electron carrier, due to depression of the oxidized versus. ASQ reduction midpoint potential, E°OX/ASQ. Perturbation of the ET site also affected the remote Bf site, whereas abrogation of Bf FAD binding accelerated chemical modification of the ET flavin. In the Bf site, removal of the positive charge impaired binding of FAD or AMP, resulting in unstable protein. Based on pH dependence, we propose that the Bf site Arg interacts with the phosphate(s) of Bf FAD or AMP, bridging the domain interface via a conserved peptide loop ("zipper") and favoring nucleotide binding. We further propose a model that rationalizes conservation of the Bf site Arg even in non-Bf ETFs, as well as AMP's stabilizing role in the latter, and provides a mechanism for coupling Bf flavin redox changes to domain-scale motion. Electron transfer flavoproteins (ETFs) are heterodimeric proteins whose fold is described by three domains (Fig. 1). The EtfA monomer includes domains I and II while domain III derives from the EtfB monomer. Domain II has been shown to reorient by some 80° relative to the base comprised of domains I and III (1Toogood H.S. Leys D. Scrutton N.S. Dynamics driving function : New insights from electron transferring flavoproteins and partner complexes.FEBS J. 2007; 274: 5481-5504Crossref PubMed Scopus (88) Google Scholar, 2Toogood H.S. van Thiel A. Scrutton N.S. Leys D. Stabilization of non-productive conformations underpins rapid electron transfer to electron transferring flavoprotein.J. Biol. Chem. 2005; 280: 30361-30366Abstract Full Text Full Text PDF PubMed Scopus (28) Google Scholar), and this conformational change has been proposed to gate electron transfer between the flavins of Bf ETF in the course of turnover (Fig. S1 and (3Demmer J.K. Bertsch J. Oppinger C. Wohlers H. Kayastha K. Demmer U. Ermler U. Muller V. Molecular basis of the flavin-based electron-bifurcating caffeyl-CoA reductase reaction.FEBS Lett. 2018; 592: 332-342Crossref PubMed Scopus (18) Google Scholar, 4Demmer J.K. Chowdhury N.P. Selmer T. Ermler U. Buckel W. The semiquinone swing in the bifurcating electron transferring flavoprotein/butyryl-coA dehydrogenase complex from Clostridium difficile.Nat. Commun. 2017; 8: 1577Crossref PubMed Scopus (53) Google Scholar)). The canonical ETFs were discovered first, in mitochondria, and employ a single FAD to mediate single-electron transfer from client CoA dehydrogenases to the quinone pool via an ETF quinone oxidoreductase, at relatively high reduction midpoint potentials (E°s) (5Thorpe C. Electron-Transferring flavoproteins.in: Müller F. Chemistry and Biochemistry of Flavoenzymes. CRC press, Boca Raton FL1991: 471-486Google Scholar, 6Jones M. Basran J. Sutcliffe M.J. Grossmann J.G. Scrutton S., N. X-ray scattering studies of Methylophilus methylotrophus (sp W(3)A(1)) electron-transferring flavoprotein - evidence for multiple conformational states and an induced fit mechanism for assembly with trimethylamine dehydrogenase.J. Biol. Chem. 2000; 275: 21349-21354Abstract Full Text Full Text PDF PubMed Scopus (18) Google Scholar, 7Roberts D.L. Frerman F.E. Kim J.J. Three-dimensional structure of human electron transfer flavoprotein to 2.1-A resolution.Proc. Natl. Acad. Sci. U. S. A. 1996; 93: 14355-14360Crossref PubMed Scopus (148) Google Scholar, 8Roberts D.L. Salazar D. Fulmer J.P. Frerman F.E. Kim J.J. Crystal structure of paracoccus denitrificans electron transfer flavoprotein: Structural and electrostatic analysis of a conserved flavin binding domain.Biochemistry. 1999; 38: 1977-1989Crossref PubMed Scopus (56) Google Scholar, 9Burgess S.G. Messiha H.L. Katona G. Rigby S.E.J. Leys D. Scrutton N.S. Probing the dynamic interface between trimethylamine dehydrogenase (TMADH) and electron transferring flavoprotein (ETF) in the TMADH-2ETF complex: Role of the Arg-α237 (ETF) and Tyr-442 (TMADH) residue pair.Biochemistry. 2008; 47: 5168-5181Crossref PubMed Scopus (9) Google Scholar, 10Dwyer T.M. Zhang L. Muller M. Marrugo F. Frerman F.E. The functions of the flavin contact residues αArg249 and βTyr16, in human electron transfer flavoprotein.Biochim. Biophys. Acta. 1999; 1433: 139-152Crossref PubMed Scopus (17) Google Scholar, 11Watmough N.J. Frerman F.E. The electron transfer flavoprotein: Ubiquinone oxidoreductases.Biochim. Biophys. Acta. 2010; 1797: 1910-1916Crossref PubMed Scopus (158) Google Scholar). More recently, research has focused on a second clade of ETFs containing two FADs (12Sato K. Nishina Y. Shiga K. Purification of electron-transferring flavoprotein from Megasphaera elsdenii and binding of additional FAD with an unusual absorption spectrum.J. Biochem. 2003; 134: 719-729Crossref PubMed Scopus (30) Google Scholar) and mediating electron bifurcation (13Garcia Costas A.M. Poudel S. Miller A.-F. J S.G. Ledbetter R.N. Fixen K. Seefeldt L.C. Adams M.W. Harwood C.S. Boyd E.S. Peters J.W. Defining electron bifurcation in the electron transferring flavoprotein family.J. Bacteriol. 2017; 199e00440-17Crossref PubMed Scopus (35) Google Scholar, 14Sato K. Nishina Y. Shiga K. Interaction between NADH and electron-transferring flavoprotein from Megasphaera elsdenii.J. Biochem. 2013; 153: 565-572Crossref PubMed Scopus (23) Google Scholar, 15Herrmann G. Jayamani E. Mai G. Buckel W. Energy conservation via electron-transferring flavoprotein in anaerobic bacteria.J. Bacteriol. 2008; 190: 784-791Crossref PubMed Scopus (283) Google Scholar). In this, NADH donates a pair of electrons to the ETF-mediated bifurcation reaction, and in this case, a client CoA dehydrogenase may be a high-potential acceptor, of one electron. This exothermic electron transfer (ET) reaction is used to drive endothermic transfer of the other electron to a lower potential (more reducing) acceptor: ferredoxin or flavodoxin semiquinone. The Bf ETFs that support nitrogen fixation were named FixAB on this basis (8Roberts D.L. Salazar D. Fulmer J.P. Frerman F.E. Kim J.J. Crystal structure of paracoccus denitrificans electron transfer flavoprotein: Structural and electrostatic analysis of a conserved flavin binding domain.Biochemistry. 1999; 38: 1977-1989Crossref PubMed Scopus (56) Google Scholar, 13Garcia Costas A.M. Poudel S. Miller A.-F. J S.G. Ledbetter R.N. Fixen K. Seefeldt L.C. Adams M.W. Harwood C.S. Boyd E.S. Peters J.W. Defining electron bifurcation in the electron transferring flavoprotein family.J. Bacteriol. 2017; 199e00440-17Crossref PubMed Scopus (35) Google Scholar, 16Weidenhaupt M. Rossi P. Beck C. Fischer H.-M. Hennecke H. Bradyrhizobium japonicum possesses two discrete sets of electron transfer flavoprotein genes:fixA, fixB and etfS, etfL.Arch. Microbiol. 1996; 165: 169-178PubMed Google Scholar, 17Ledbetter R.N. Garcia Costas A.M. Lubner C.E. Mulder D.E. Tokmina-Lukaszewska M. Artz J.H. Patterson A. Magnuson T.S. Jay Z.J. Duan H.D. Miller J. Plunkett M.H. Hoben J.P. Barney B.M. Carlson R.P. et al.The electron bifurcating FixABCX protein complex from Azotobacter vinelandii: Generation of low-potential reducing equivalents for nitrogenase catalysis.Biochemistry. 2017; 56: 4177-4190Crossref PubMed Scopus (82) Google Scholar) and employ a quinone reductase comprised of subunits FixC and FixX as an exergonic acceptor. The FAD that accepts a pair of electrons (2e) from NADH and dispenses them to separate paths is called the bifurcating FAD (Bf FAD, green in Fig. 1), whereas the flavin analogous to that of canonical ETFs is called the ET FAD (yellow in Fig. 1). It is critical that the ET FAD mediates single electron (1e) transfer only, as exergonic transfer of both electrons would dissipate the reducing energy inherent in NADH. The flavin moiety of the Bf FAD is bound between domains I and III while its AMP portion replaces the AMP of canonical ETFs (18Chowdhury N.P. Mowafy A.M. Demmer J.K. Upadhyay V. Koelzer S. Jayamani E. Kahnt J. Hornung M. Demmer U. Ermler U. Buckel W. Studies on the mechanism of electron bifurcation catalyzed by electron transferring flavoprotein (Etf) and butyryl-CoA dehydrogenase (Bcd) of Acidaminococcus fermentans.J. Biol. Chem. 2014; 289: 5145-5157Abstract Full Text Full Text PDF PubMed Scopus (78) Google Scholar). Working with the Bf ETF of Rhodopseudomonas palustris (RpaETF), Duan et al. (19Duan H.D. Lubner C.E. Tokmina-Lukaszewska M. Gauss G.H. Bothner B. King P.W. Peters J.W. Miller A.F. Distinct flavin properties underlie flavin-based electron bifurcation within a novel electron-transferring flavoprotein FixAB from Rhodopseudomonas palustris.J. Biol. Chem. 2018; 293: 4688-4701Abstract Full Text Full Text PDF PubMed Scopus (13) Google Scholar) demonstrated that the two FADs satisfy the thermodynamic requirements for electron bifurcation. Specifically, the ET FAD should have two high-potential 1e E°s suiting it to transfer electrons one at a time between the Bf FAD and the FixCX (Fig. 1). Thus, the ET FAD should be able to adopt a semiquinone (SQ) state and cycle between it and either the oxidized (OX) state or the fully reduced hydroquinone (HQ) state. Indeed, the ET FAD was observed to accumulate in the anionic SQ state (ASQ) part way through reductive titrations (10Dwyer T.M. Zhang L. Muller M. Marrugo F. Frerman F.E. The functions of the flavin contact residues αArg249 and βTyr16, in human electron transfer flavoprotein.Biochim. Biophys. Acta. 1999; 1433: 139-152Crossref PubMed Scopus (17) Google Scholar, 14Sato K. Nishina Y. Shiga K. Interaction between NADH and electron-transferring flavoprotein from Megasphaera elsdenii.J. Biochem. 2013; 153: 565-572Crossref PubMed Scopus (23) Google Scholar, 20Yang K.Y. Swenson R.P. Modulation of the redox properties of the flavin cofactor through hydrogen-bonding interactions with the N(5) atom: Role of alpha Ser254 in the electron-transfer flavoprotein from the methylotrophic bacterium W3A1.Biochemistry. 2007; 46: 2289-2297Crossref PubMed Scopus (23) Google Scholar). In contrast, the Bf FAD should have a single 2e couple with a lower E°, between those of NADH and the ET FAD (21Nitschke W. Russell M.J. Redox bifurcations: Mechanisms and importance to life now, and at its origin.Bioessays. 2012; 34: 106-109Crossref PubMed Scopus (94) Google Scholar). This too was documented in Bf ETFs from R. palustris (19Duan H.D. Lubner C.E. Tokmina-Lukaszewska M. Gauss G.H. Bothner B. King P.W. Peters J.W. Miller A.F. Distinct flavin properties underlie flavin-based electron bifurcation within a novel electron-transferring flavoprotein FixAB from Rhodopseudomonas palustris.J. Biol. Chem. 2018; 293: 4688-4701Abstract Full Text Full Text PDF PubMed Scopus (13) Google Scholar), Megasphaera elsdenii (MelETF) (14Sato K. Nishina Y. Shiga K. Interaction between NADH and electron-transferring flavoprotein from Megasphaera elsdenii.J. Biochem. 2013; 153: 565-572Crossref PubMed Scopus (23) Google Scholar), Pyrobaculum aerophilum (PaeETF) (22Schut G.J. Mohamed-Raseek N.R. Tokmina-Lukaszewska M. Mulder D.E. Nguyen D.M.N. Lipscomb G.L. Hoben J.P. Patterson A. Lubner C.E. King P.W. Peters J.W. Bothner B. Miller A.F. Adams M.W.W. The catalytic mechanism of electron bifurcating electron transfer flavoproteins (ETFs) involves an intermediary complex with NAD+.J. Biol. Chem. 2019; 294: 3271-3283Abstract Full Text Full Text PDF PubMed Scopus (22) Google Scholar) and Acidaminococcus fermentans (AfeETF) (23Sucharitakul J. Buttranon S. Wongnate T. Chowdhury N.P. Prongjit M. Buckel W. Chaiyen P. Modulations of the reduction potentials of flavin-based electron bifurcation complexes and semiquinone stabilities are key to control directional electron flow.FEBS J. 2020; 288: 1008-1026Crossref PubMed Scopus (11) Google Scholar). The structural identities of the two FADs were deduced based on the analogy of one with canonical ETFs' FAD, in conjunction with binding of NADH near the other (18Chowdhury N.P. Mowafy A.M. Demmer J.K. Upadhyay V. Koelzer S. Jayamani E. Kahnt J. Hornung M. Demmer U. Ermler U. Buckel W. Studies on the mechanism of electron bifurcation catalyzed by electron transferring flavoprotein (Etf) and butyryl-CoA dehydrogenase (Bcd) of Acidaminococcus fermentans.J. Biol. Chem. 2014; 289: 5145-5157Abstract Full Text Full Text PDF PubMed Scopus (78) Google Scholar). Experimental tests characterized flavin known to reside in domain II, based on mutagenesis to replace threonine 94 and 97 that hydrogen bond (H-bond) to ribose of the FAD between domains I and III (T94,97A-RpaETF), as well as computation to model each flavin's visible spectrum based on its environment in the protein (24Mohamed-Raseek N. Duan H.D. Mroginski M.A. Miller A.F. Spectroscopic, thermodynamic and computational evidence of the locations of the FADs in the nitrogen fixation-associated electron transfer flavoprotein.Chem. Sci. 2019; 10: 7762-7772Crossref PubMed Google Scholar). These site-specific approaches confirmed the structural models in assigning functions to each of the flavins in RpaETF (Fig. 1). Given the contrasting reactivities of the two flavins, it was expected that their two binding sites would be different too, as noncovalent interactions with their protein surroundings are believed to modulate the reactivity of bound flavins (20Yang K.Y. Swenson R.P. Modulation of the redox properties of the flavin cofactor through hydrogen-bonding interactions with the N(5) atom: Role of alpha Ser254 in the electron-transfer flavoprotein from the methylotrophic bacterium W3A1.Biochemistry. 2007; 46: 2289-2297Crossref PubMed Scopus (23) Google Scholar, 25Massey V. Hemmerich P. Active site probes of flavoproteins.Biochem. Soc. Trans. 1980; 8: 246-257Crossref PubMed Scopus (278) Google Scholar, 26Lostao A. Gomez-Moreno C. Mayhew S.G. Sancho J. Differential stabilization of the three FMN redox forms by tyrosine 94 and tryptophan 57 in flavodoxin from Anabaena and its influence on the redox potentials.Biochemistry. 1997; 36: 14334-14344Crossref PubMed Scopus (81) Google Scholar, 27Bradley L.H. Swenson R.P. Role of hydrogen bonding interactions to N(3)H of the flavin mononucleotide cofactor in the modulation of the redox potentials of the Clostridium beijerinckii flavodoxin.Biochemistry. 2001; 40: 8686-8695Crossref PubMed Scopus (28) Google Scholar). There are, indeed, differences. However, from an electrostatic standpoint, both sites are dominated by an Arg residue that is highly conserved. Since the Arg residues of interest both derive from the A subunit of ETF, we omit the chain specifier and name them by their residue numbers alone. Residue 273 in the ET site is conserved as Arg in 202 of 216 sequences of Bf ETFs, and as lysine (Lys) in ten of them, whereas residue 165 in the Bf site is Arg in 214 of 216 Bf ETFs (and Lys in 1) (13Garcia Costas A.M. Poudel S. Miller A.-F. J S.G. Ledbetter R.N. Fixen K. Seefeldt L.C. Adams M.W. Harwood C.S. Boyd E.S. Peters J.W. Defining electron bifurcation in the electron transferring flavoprotein family.J. Bacteriol. 2017; 199e00440-17Crossref PubMed Scopus (35) Google Scholar). In each case, the Arg is the only charged residue close enough to interact directly with the flavin. In the case of the ET site, one can rationalize the high E° of the OX/ASQ couple (E°OX/ASQ) in terms of a favorable interaction between the ASQ formed and the nearby Arg-273 side chain (10Dwyer T.M. Zhang L. Muller M. Marrugo F. Frerman F.E. The functions of the flavin contact residues αArg249 and βTyr16, in human electron transfer flavoprotein.Biochim. Biophys. Acta. 1999; 1433: 139-152Crossref PubMed Scopus (17) Google Scholar, 28Talfournier F. Munro A.W. Basran J. Sutcliffe M.J. Daff S. Chapman S.K. Scrutton N.S. Alpha Arg-237 in Methylophilus methylotrophus (sp. W3A1) electron-transferring flavoprotein affords approximately 200-millivolt stabilization of the FAD anionic semiquinone and a kinetic block on full reduction to the dihydroquinone.J. Biol. Chem. 2001; 276: 20190-20196Abstract Full Text Full Text PDF PubMed Scopus (28) Google Scholar). However, it is difficult to rationalize the unstable ASQ in Bf site with the nearby side chain of Arg-165. Not only is the ASQ thermodynamically suppressed, in that it is not seen as an intermediate between OX and HQ in reductive titrations, but its instability (high energy) is understood to be crucial to the Bf flavin's ability to reduce a low-E° acceptor (21Nitschke W. Russell M.J. Redox bifurcations: Mechanisms and importance to life now, and at its origin.Bioessays. 2012; 34: 106-109Crossref PubMed Scopus (94) Google Scholar, 29Peters J.W. Miller A.F. Jones A.K. King P.W. Adams M.W. Electron bifurcation.Curr. Opin. Chem. Biol. 2016; 31: 146-152Crossref PubMed Scopus (77) Google Scholar). However, we would expect it to be stabilized by a nearby Arg. To elucidate the paradox of contrasting reactivities stemming from two flavins presumed to be chemically identical and both in contact with a conserved Arg, we replaced each Arg and characterized the resulting variants' stability, nucleotide content, and redox reactivity. Ours are the first biophysical characterizations to our knowledge of Bf ETFs incorporating amino acid substitutions in the Bf site. Our strategy rests on the firm foundation provided by variants of canonical ETFs containing substitutions in the ET site (9Burgess S.G. Messiha H.L. Katona G. Rigby S.E.J. Leys D. Scrutton N.S. Probing the dynamic interface between trimethylamine dehydrogenase (TMADH) and electron transferring flavoprotein (ETF) in the TMADH-2ETF complex: Role of the Arg-α237 (ETF) and Tyr-442 (TMADH) residue pair.Biochemistry. 2008; 47: 5168-5181Crossref PubMed Scopus (9) Google Scholar, 10Dwyer T.M. Zhang L. Muller M. Marrugo F. Frerman F.E. The functions of the flavin contact residues αArg249 and βTyr16, in human electron transfer flavoprotein.Biochim. Biophys. Acta. 1999; 1433: 139-152Crossref PubMed Scopus (17) Google Scholar, 28Talfournier F. Munro A.W. Basran J. Sutcliffe M.J. Daff S. Chapman S.K. Scrutton N.S. Alpha Arg-237 in Methylophilus methylotrophus (sp. W3A1) electron-transferring flavoprotein affords approximately 200-millivolt stabilization of the FAD anionic semiquinone and a kinetic block on full reduction to the dihydroquinone.J. Biol. Chem. 2001; 276: 20190-20196Abstract Full Text Full Text PDF PubMed Scopus (28) Google Scholar). However, our studies extend the approach to the Arg of the Bf site and demonstrate that the status of each flavin site affects the other. These long-range effects raise the possibility of conformational coupling. Indeed, a remarkable 80° rotation of domain II relative to the domain I⋅III base has been documented crystallographically and is proposed to gate electron transfer in Bf ETFs (3Demmer J.K. Bertsch J. Oppinger C. Wohlers H. Kayastha K. Demmer U. Ermler U. Muller V. Molecular basis of the flavin-based electron-bifurcating caffeyl-CoA reductase reaction.FEBS Lett. 2018; 592: 332-342Crossref PubMed Scopus (18) Google Scholar, 4Demmer J.K. Chowdhury N.P. Selmer T. Ermler U. Buckel W. The semiquinone swing in the bifurcating electron transferring flavoprotein/butyryl-coA dehydrogenase complex from Clostridium difficile.Nat. Commun. 2017; 8: 1577Crossref PubMed Scopus (53) Google Scholar) and engage partner proteins (1Toogood H.S. Leys D. Scrutton N.S. Dynamics driving function : New insights from electron transferring flavoproteins and partner complexes.FEBS J. 2007; 274: 5481-5504Crossref PubMed Scopus (88) Google Scholar). With this in mind, we discuss a mechanism suggested by our finding that mutation of Arg-165 appears to abrogate binding not only of FAD, but even AMP in the Bf site. We propose that Arg-165 establishes a network of H-bonds reaching between domains I and III via a conserved peptide loop that bridges between Arg-165 and the phosphate(s) of AMP/FAD. Such a network stabilizing the dimer interface would explain the conservation of Arg-165 even among non-Bf ETFs, as well as retention of AMP as a stabilizer of the quaternary structure (30Sato K. Nishima Y. Shiga K. In vitro assembly of FAD, AMP, and the two subunits of electron-transferring flavoprotein: An important role of AMP related with the conformational change of the apoprotein.J. Biochem. 1997; 121: 477-486Crossref PubMed Scopus (11) Google Scholar). It also provides an elegant mechanism by which the oxidation state of the Bf flavin could exert conformational consequences coupling the domain rotation to electron movements. Considering the ET FAD's unusually stable ASQ state and precedent in canonical ETFs (10Dwyer T.M. Zhang L. Muller M. Marrugo F. Frerman F.E. The functions of the flavin contact residues αArg249 and βTyr16, in human electron transfer flavoprotein.Biochim. Biophys. Acta. 1999; 1433: 139-152Crossref PubMed Scopus (17) Google Scholar, 28Talfournier F. Munro A.W. Basran J. Sutcliffe M.J. Daff S. Chapman S.K. Scrutton N.S. Alpha Arg-237 in Methylophilus methylotrophus (sp. W3A1) electron-transferring flavoprotein affords approximately 200-millivolt stabilization of the FAD anionic semiquinone and a kinetic block on full reduction to the dihydroquinone.J. Biol. Chem. 2001; 276: 20190-20196Abstract Full Text Full Text PDF PubMed Scopus (28) Google Scholar), it was inferred that a crucial feature of the ET site would be the conserved Arg-273 that should be positively charged under physiological conditions. However, the Bf ETFs demonstrate that conservation of a nearby Arg does not guarantee a stable ASQ state, as the bifurcating site also contains a conserved Arg, yet suppresses the Bf flavin's SQ states. Thus, the effect appears more nuanced than the identity of the residue, and we expect that the nature of the interaction between Arg and flavin is different in the Bf site than in the ET site. Indeed, Arg-273 is seen in crystal structures to form a π-π stacking interaction with ET FAD (4Demmer J.K. Chowdhury N.P. Selmer T. Ermler U. Buckel W. The semiquinone swing in the bifurcating electron transferring flavoprotein/butyryl-coA dehydrogenase complex from Clostridium difficile.Nat. Commun. 2017; 8: 1577Crossref PubMed Scopus (53) Google Scholar, 18Chowdhury N.P. Mowafy A.M. Demmer J.K. Upadhyay V. Koelzer S. Jayamani E. Kahnt J. Hornung M. Demmer U. Ermler U. Buckel W. Studies on the mechanism of electron bifurcation catalyzed by electron transferring flavoprotein (Etf) and butyryl-CoA dehydrogenase (Bcd) of Acidaminococcus fermentans.J. Biol. Chem. 2014; 289: 5145-5157Abstract Full Text Full Text PDF PubMed Scopus (78) Google Scholar) whereas Arg-165 appears to form bidentate H-bonding interactions with the N5 of Bf FAD and a peptide loop that flanks it (Fig. 1C) (31Kayastha K. Vitt S. Buckel W. Ermler U. Flavins in the electron bifurcation process.Arch. Biochem. Biophys. 2021; 701: 108796Crossref PubMed Scopus (7) Google Scholar). To test the natures of the interactions, we generated variants, which remove the positive charge (replacement with alanine or glutamine, Ala or Gln) or remove bidentate H-bonding (replacement with Lys). We also sought to make the charge tunable via modulation of pH, by replacing Arg with histidine (His). Amino acid substitutions were made in each of the two flavin-binding sites separately. The variants affecting Arg-273 in the ET site were expressed in soluble form and purified. The yields of 20 to 25 mg per liter of culture were comparable to that of the WT indicating similarly stable and soluble proteins. Consistent with this, these variants all contained two FADs per heterodimer after purification including exposure to 1 mM FAD at the time of cell lysis, to repopulate any FAD-binding sites having good affinity (Table 1). Of the variants affecting the Bf site, R165K-RpaETF behaved like the WT. (Throughout, the R165K notation is used to indicate the variant in which Lys replaces Arg at position 165 of the EtfA.) However, variants lacking a cation in the Bf site displayed lowered affinity for FAD. The R165H-RpaETF could be purified with close to one bound FAD, whereas R165Q-RpaETF could only be purified in low yield with less than one FAD bound at pH 8 (below). R165A-RpaETF was expressed but partitioned into the insoluble fraction and produced colorless, sparingly stable protein in minute yield.Table 1Flavin contents and thermal stabilitiesRpaETF variantFAD content/dimerTm (°C)aTm values were measured on 17 to 30 μM ETF in 20 mM working buffer with 200 mM KCl and 10% (w/v) glycerol at pH 8.WT2.00 ± 0.00bStandard deviations of 0.00 indicate that the two independent trials gave the same value.46 ± 2R273A2.04 ± 0.0644 ± 0R273H2.03 ± 0.0444 ± 0R273K2.09 ± 0.0744 ± 0R165K2.00 ± 0.0044 ± 0R165H0.9 ± 0.140 ± 2a Tm values were measured on 17 to 30 μM ETF in 20 mM working buffer with 200 mM KCl and 10% (w/v) glycerol at pH 8.b Standard deviations of 0.00 indicate that the two independent trials gave the same value. Open table in a new tab The Tm values marking the midpoints of thermal denaturation at pH 8 displayed the same trends. Protein secondary structure was monitored via far-UV CD as the temperature was raised. All variants studied displayed similar secondary structure content at 25 °C. The variants that were soluble and readily purified all had Tm values only slightly lower than that of WT. The exception was the R165H-RpaETF for which the Tm was lower. To learn about the environment sensed by the flavins in the modified sites, we compared visible spectra of the variants with those of WT. The absorption bands of the two oxidized flavins overlap almost completely, so for better insight, we exploited the fact that in all variants the ET flavin underwent full reduction before the Bf flavin began to reduce, so a spectrum collected halfway through a reductive titration retained the signature of OX Bf flavin and ET flavin HQ. The difference spectrum from the first half of a titration therefore reflects ET FAD OX minus HQ, and the corresponding difference spectrum for Bf FAD was obtained from the second half of the titration (see Experimental procedures and Fig. S2). We assumed that the two HQ spectra present in the endpoint spectrum are very similar, and in any case they are weak compared with the OX spectra, especially at longer wavelengths. Therefore, we used the end-point spectrum divided by two as a proxy for either flavin's HQ. Addition of this spectrum to the above difference spectra yielded the deduced OX spectra for the ET flavin and Bf flavin. These are compared in Figure 2 and Table 2. The R165H-RpaETF and R165Q-RpaETF variants displayed several distinct properties, so they are discussed together in later sections.Table 2Comparison of absorption maxima of individual flavins in each variant, at pH 8RpaETF variantET FADBf FADBand I (near 450 nm)(±2 nm)Band II (near 380 nm)(±2 nm)Band height ratio II/IBand I (near 450 nm)(±2 nm)Band II (near 380 nm)(±2 nm)Band height ratio II/IWT4483941.044543740.98R273K4503961.064543740.99R273A4463921.054543740.99R273H4463941.054543740.96R165K4483961.074603781.09R165H4523861.01N/AN/AN/A Open table in a new tab In all our variants except R165K-RpaETF, the Bf FAD displayed vibrational features in band I, and in all cases, the two bands were well separated, absorbing around 450 and 370 nm, respectively, with comparable band heights, usually favoring band I. On the other hand, for ET FAD, the separation between bands I and II was smaller, producing a shallower dip between them (band II is shifted to 386–392 nm), and the amplitude of band II was higher than or equal to that of band I. Thus the red-shifted band II position of ET FAD in WT-RpaETF (394 versus. free FAD at 374 n
Referência(s)