Crystal Structure of Paracoccus denitrificans Electron Transfer Flavoprotein:  Structural and Electrostatic Analysis of a Conserved Flavin Binding Domain^,

Abstract

The crystal structure of electron transfer flavoprotein (ETF) from Paracoccus denitrificans was determined and refined to an R-factor of 19.3% at 2.6 A resolution. The overall fold is identical to that of the human enzyme, with the exception of a single loop region. Like the human structure, the structure of the P. denitrificans ETF is comprised of three distinct domains, two contributed by the alpha-subunit and the third from the beta-subunit. Close analysis of the structure reveals that the loop containing betaI63 is in part responsible for conferring the high specificity of AMP binding by the ETF protein. Using the sequence and structures of the human and P. denitrificans enzymes as models, a detailed sequence alignment has been constructed for several members of the ETF family, including sequences derived for the putative FixA and FixB proteins. From this alignment, it is evident that in all members of the ETF family the residues located in the immediate vicinity of the FAD cofactor are identical, with the exception of the substitution of serine and leucine residues in the W3A1 ETF protein for the human residues alphaT266 and betaY16, respectively. Mapping of ionic differences between the human and P. denitrificans ETF onto the structure identifies a surface that is electrostatically very similar between the two proteins, thus supporting a previous docking model between human ETF and pig medium-chain acyl-CoA dehydrogenase (MCAD). Analysis of the ionic strength dependence of the electron transfer reaction between either human or P. denitrificans ETF and MCAD demonstrates that the human ETF functions optimally at low ( approximately 10 mequiv) ionic strength, while P. denitrificans ETF is a better electron acceptor at higher (>75 mequiv) ionic strength. This suggests that the electrostatic surface potential of the two proteins is very different and is consistent with the difference in isoelectric points between the proteins. Analysis of the electrostatic potentials of the human and P. denitrificans ETFs reveals that the P. denitrificans ETF is more negatively charged. This excess negative charge may contribute to the difference in redox potentials between the two ETF flavoproteins and suggests an explanation for the opposing ionic strength dependencies for the reaction of MCAD with the two ETFs. Furthermore, by analysis of a model of the previously described human-P. denitrificans chimeric ETF protein, it is possible to identify one region of ETF that participates in docking with ETF-ubiquinone oxidoreductase, the physiological electron acceptor for ETF.

Dissection of the Caffeate Respiratory Chain in the Acetogen Acetobacterium woodii : Identification of an Rnf-Type NADH Dehydrogenase as a Potential Coupling Site

“…Etf proteins are heterodimers of EtfA and EtfB and contain noncovalently bound flavin adenine dinucleotide (FAD). The crystal structure of EtfA of Paracoccus denitrificans was solved, and the FAD binding site was identified in the structure in the conserved C terminus (44,45). The Q240, V241, Q243, T244, Q263, and H264 residues that interact with the isoalloxazine ring of the FAD in P. denitrificans are fully conserved in EtfA Aw .…”

“…FAD's ribityl and ADP moiety is coordinated by 10 additional residues, 6 of which (S226, R227, S259, N278, K279, and D296) are also conserved in EtfA Aw . EtfB contains the binding site for a second cofactor, AMP (44,45). Two of the residues shown to be involved in AMP binding (G120 and A123 in P. denitrificans) are also conserved in EtfB Aw .…”

Protein Dynamics Enhance Electronic Coupling in Electron Transfer Complexes

“…Simulated X-ray Scattering Profiles-The x-ray structures of human and P. denitrificans ETFs have been solved previously (14,15). Using these atomic coordinates, simulated x-ray scattering profiles were generated for solvated structures of both human and P. denitrificans ETFs and fitted against the experimental data for each protein, respectively (Fig.…”

“…The catalytic and redox properties of the two proteins are very similar (12,13). Crystal structures have been determined for both ETFs at resolutions of 2.1 and 2.6 Å, respectively (14,15). These reveal that the overall fold for both proteins is identical with the exception of a single loop region.…”

αArg-237 in Methylophilus methylotrophus (sp. W3A1) Electron-transferring Flavoprotein Affords ∼200-Millivolt Stabilization of the FAD Anionic Semiquinone and a Kinetic Block on Full Reduction to the Dihydroquinone

“…All ETFs possess one equivalent of non-covalently bound FAD per ETF heterodimer, except the ETF from Megasphaera elsdenii, which contains 2 equivalents of FAD per dimer (8). It has been shown that AMP (1 equivalent) is associated with the housekeeping ETFs from pigs (9), humans (10), and Paracoccus denitrificans (11) and with the specialized ETF from Methylophilus methylotrophus (12).…”