Spectroscopic evidence for direct flavin-flavin contact in a bifurcating electron transfer flavoprotein
2020; Elsevier BV; Volume: 295; Issue: 36 Linguagem: Inglês
10.1074/jbc.ra120.013174
ISSN1083-351X
AutoresH. Diessel Duan, Nishya Mohamed‐Raseek, Anne‐Frances Miller,
Tópico(s)Photoreceptor and optogenetics research
ResumoA remarkable charge transfer (CT) band is described in the bifurcating electron transfer flavoprotein (Bf-ETF) from Rhodopseudomonas palustris (RpaETF). RpaETF contains two FADs that play contrasting roles in electron bifurcation. The Bf-FAD accepts electrons pairwise from NADH, directs one to a lower-reduction midpoint potential (E°) carrier, and the other to the higher-E° electron transfer FAD (ET-FAD). Previous work noted that a CT band at 726 nm formed when ET-FAD was reduced and Bf-FAD was oxidized, suggesting that both flavins participate. However, existing crystal structures place them too far apart to interact directly. We present biochemical experiments addressing this conundrum and elucidating the nature of this CT species. We observed that RpaETF missing either FAD lacked the 726 nm band. Site-directed mutagenesis near either FAD produced altered yields of the CT species, supporting involvement of both flavins. The residue substitutions did not alter the absorption maximum of the signal, ruling out contributions from residue orbitals. Instead, we propose that the residue identities modulate the population of a protein conformation that brings the ET-flavin and Bf-flavin into direct contact, explaining the 726 nm band based on a CT complex of reduced ET-FAD and oxidized Bf-FAD. This is corroborated by persistence of the 726 nm species during gentle protein denaturation and simple density functional theory calculations of flavin dimers. Although such a CT complex has been demonstrated for free flavins, this is the first observation of such, to our knowledge, in an enzyme. Thus, Bf-ETFs may optimize electron transfer efficiency by enabling direct flavin-flavin contact. A remarkable charge transfer (CT) band is described in the bifurcating electron transfer flavoprotein (Bf-ETF) from Rhodopseudomonas palustris (RpaETF). RpaETF contains two FADs that play contrasting roles in electron bifurcation. The Bf-FAD accepts electrons pairwise from NADH, directs one to a lower-reduction midpoint potential (E°) carrier, and the other to the higher-E° electron transfer FAD (ET-FAD). Previous work noted that a CT band at 726 nm formed when ET-FAD was reduced and Bf-FAD was oxidized, suggesting that both flavins participate. However, existing crystal structures place them too far apart to interact directly. We present biochemical experiments addressing this conundrum and elucidating the nature of this CT species. We observed that RpaETF missing either FAD lacked the 726 nm band. Site-directed mutagenesis near either FAD produced altered yields of the CT species, supporting involvement of both flavins. The residue substitutions did not alter the absorption maximum of the signal, ruling out contributions from residue orbitals. Instead, we propose that the residue identities modulate the population of a protein conformation that brings the ET-flavin and Bf-flavin into direct contact, explaining the 726 nm band based on a CT complex of reduced ET-FAD and oxidized Bf-FAD. This is corroborated by persistence of the 726 nm species during gentle protein denaturation and simple density functional theory calculations of flavin dimers. Although such a CT complex has been demonstrated for free flavins, this is the first observation of such, to our knowledge, in an enzyme. Thus, Bf-ETFs may optimize electron transfer efficiency by enabling direct flavin-flavin contact. Electron transfer is central to energy metabolism in all kingdoms of life. Of the redox cofactors used, FMN and FAD are among the most versatile (1Massey V. The chemical and biochemical versatility of riboflavin.Biochem. Soc. Trans. 2000; 28: 283-29610.1042/0300-5127:0280283Crossref PubMed Google Scholar, 2Mansoorabadi S.O. Thibodeaux C.J. Liu H.W. 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Qualitatively similar behavior was observed with NADH as the reductant, although maximization and decay of the signal occurred at somewhat higher NADH concentrations, suggesting that stoichiometric additions of NADH do not fall in the tight-binding regime (see below). However, the 726 nm species itself does not appear to be a transient intermediate as it was stable over days under inert atmosphere. The band is seen to extend to long wavelengths as for other flavin CT bands, but it displays apparent structure (Fig. 2B). Whereas vibrational structure is evident in absorption bands of individual flavins, it is generally not observable on the broader supramolecular CT transitions (47Xiang Q. Guo J. Xu J. Ding S. Li Z. Li G. Phan H. Gu Y. Dang Y. Xu Z. Gong Z. Hu W. Zeng Z. Wu J.F. Sun Z. Stable olympicenyl radicals and their π-dimers.J. Am. Chem. Soc. 2020; 142 (32456437): 11022-1103110.1021/jacs.0c02287Crossref PubMed Scopus (1) Google Scholar). We speculate that the protein environment may suppress movement of one partner relative to the other that might otherwise be associated with the transition. The 726 nm band could also be formed in oxidative titrations (Fig. 2C). When NAD+ was used to oxidize RpaETF that had been fully reduced by prior treatment with dithionite, the CT band at 726 nm grew initially, concurrent with the appearance of spectral features of OX flavin (Fig. 2C). Upon the addition of excess NAD+, it was even possible to decrease the amount of 726 nm species present as more flavin was converted to OX (below). Therefore, the 726 nm species represents a state function rather than an intermediate of a particular path. The 726 nm band appeared at 726 ± 2 nm at pH 8 and pH 9; however, the amplitude of the 726 nm band was 4.5-fold larger at pH 9, in titrations with NADH. Thus, it does not appear to reflect the conjugate acid of an acid/base equilibrium, and we rule out the NSQ state of a flavin, although that of 8-formyl flavin can absorb in this range (48Augustin P. Toplak M. Fuchs K. Gerstmann E.C. Prassl R. Winkler A. Macheroux P. Oxidation of the FAD cofactor to the 8-formyl-derivative in human electron-transferring flavoprotein.J. Biol. Chem. 2018; 293 (29301933): 2829-284010.1074/jbc.RA117.000846Abstract Full Text Full Text PDF PubMed Scopus (0) Google Scholar) (but see Ref. 49Lehman T.C. Thorpe C. A new form of mammalian electron transfer flavoprotein.Arch. Biochem. Biophys. 1992; 292 (1731621): 594-59910.1016/0003-9861(92)90036-VCrossref PubMed Scopus (0) Google Scholar). Thus, although the use of pH 9 incurs the risk of increased 8-formyl flavin formation (48Augustin P. Toplak M. Fuchs K. Gerstmann E.C. Prassl R. Winkler A. Macheroux P. Oxidation of the FAD cofactor to the 8-formyl-derivative in human electron-transferring flavoprotein.J. Biol. Chem. 2018; 293 (29301933): 2829-284010.1074/jbc.RA117.000846Abstract Full Text Full Text PDF PubMed Scopus (0) Google Scholar), we used pH 9 to optimize our ability to detect and quantify the 726 nm signal to elucidate the nature of the species responsible. Other work under way is testing a possible relationship between formation of the 726 nm species and accumulation of modified flavin, because the latter is also favored by higher pH and correlated with a particular conformation of the ETF (48Augustin P. Toplak M. Fuchs K. Gerstmann E.C. Prassl R. Winkler A. Macheroux P. Oxidation of the FAD cofactor to the 8-formyl-derivative in human electron-transferring flavoprotein.J. Biol. Chem. 2018; 293 (29301933): 2829-284010.1074/jbc.RA117.000846Abstract Full Text Full Text PDF PubMed Scopus (0) Google Scholar) (Fig. S1). The higher 726 nm species yield at high pH suggests that the 726 nm species is favored by deprotonation of an amino acid side chain nearby or equivalently that another state in equilibrium with the 726 nm species is disfavored by the amino acid's deprotonation. To identify candidate amino acids, one must know where the 726 nm species resides. In our previous studies, we showed that the 726 nm species correlates with the oxidation states of both ET-FAD and Bf-FAD (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 (29462786): 4688-470110.1074/jbc.RA117.000707Abstract Full Text Full Text PDF PubMed Scopus (0) Google Scholar). We therefore proposed a delocalized CT involving both flavins (50Geng J. Dornevil K. Davidson V.L. Liu A. Tryptophan-mediated charge-resonance stabilization in the bis-Fe(IV) redox state of MauG.Proc. Natl. Acad. Sci. U. S. A. 2013; 110 (23720312): 9639-964410.1073/pnas.13015441
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