| Auracyanin?Ac?is a newly identified blue copper protein that mediates the electron transfer between Alternative Complex ??AC??and reaction center in the cyclic electron transport chain of filamentous anoxygenic phototrophs.In this study,we extracted and purified the air-oxidized Rfx Ac from the photoheterotrophically grown Roseifexus castenholzii,and we illustrated the structural basis underlying its electron transferring features.Spectroscopic and enzymatic analyses demonstrated the reduction of air-oxidized Rfx Ac by the AC? upon oxidation of Menaquinol-4 or Menaquinol-7.We further determined the crystal structures of the air-oxidized and Na-dithionite-reduced Rfx Ac at 2.2 and 2.0?resolutions,respectively.The overall structure contains two?helices,eight?strands and one copper ion.In the air-oxidized structure,the copper ion is coordinated by His77,His146,Cys141,and Met151;in the dithionite-reduced structure,the two loops?loop-A and loop-B?surrounding the Cu1+binding pocket undergo minor conformational changes,and the Cu1+-S??Met151?bond length increase 0.3?.The Fourier difference map between the air-oxidized and Na-dithionite-reduced Rfx Ac indicated that the electrons accepted by Ac were transferred to Cu1+,His77,and surrounding loop-B.According to N-terminal sequencing and peptide mass fingerprinting,we found that Ac may be anchored to the membrane through a glycine-rich sequence at the N-terminus,and oscillate the copper ion binding domain in the periplasm.Additionally,a conserved hydrophobic surface near the copper binding pocket may mediate the interaction of Ac with AC? and RC in the cyclic electron transfer.Therefore,we systematically studied the conformational changes of naturally extracted Ac in the oxidation and reduction states,and revealed the structural basis and mechanism of its role in cyclic electron transfer of R.castenholzii,which will contribute to the future studies of the electron transfer mechanism. |
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