Crystal structure of Paracoccus denitrificans electron transfer flavoprotein: Structural and electrostatic analysis of a conserved flavin binding domain

The crystal structure of electron transfer flavoprotein (ETF) from Paracocc us denitrificans was determined and refined to an R-factor of 19.3% at 2.6 Angstrom resolution. The overall fold is identical to that of the human enz yme, with the exception of a single loop region. Like the human structure, the structure of the P. denitrificans ETF is comprised of three distinct do mains, two contributed by the alpha-subunit and the third from the beta-sub unit. Close analysis of the structure reveals that the loop containing beta I63 is in part responsible for conferring the high specificity of AMP bind ing 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 deri ved for the putative FixA and FixB proteins, From this alignment, it is evi dent that in all members of the ETF family the residues located in the imme diate vicinity of the FAD cofactor are identical, with the exception of the substitution of serine and leucine residues in the W3A1 ETF protein for th e human residues alpha T266 and beta Y16, respectively. Mapping of ionic di fferences between the human and P. denitrificans ETF onto the structure ide ntifies a surface that is electrostatically very similar between the two pr oteins, 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. den itrificans ETF and MCAD demonstrates that the human ETF functions optimally at low (similar to 10 mequiv) ionic strength, while P, denitrificans ETF i s a better electron acceptor at higher (>75 mequiv) ionic strength. This su ggests that the electrostatic surface potential of the two proteins is very different and is consistent with the difference in isoelectric points betw een the proteins. Analysis of the electrostatic potentials of the human and P. denitrificans ETFs reveals that the P. denitrificans ETF is more negati vely charged. This excess negative charge may contribute to the difference in redox potentials between the two ETF flavoproteins and suggests an expla nation for the opposing ionic strength dependencies for the reaction of MCA D with the two ETFs. Furthermore, by analysis of a model of the previously described human-P. denitrificans chimeric ETF protein, it is possible to id entify one region of ETF that participates in docking with ETF-ubiquinone o xidoreductase, the physiological electron acceptor for ETF.