In vivo assembly of a truncated H subunit mutant of the Rhodobacter sphaeroides photosynthetic reaction centre and direct electron transfer from the QA quinone to an electrode
2018
Jun, D. | Dhupar, H. S. | Mahmoudzadeh, A. | Duong, F. | Madden, J. D. W. | Beatty, J. T.
We address a challenge in the engineering of proteins to redirect electron transfer pathways, using the bacterial photosynthetic reaction centre (RC) pigment–protein complex. Direct electron transfer is shown to occur from the QA quinone of the Rhodobacter sphaeroides RC containing a truncated H protein and bound on the quinone side to a gold electrode. In previous reports of binding to the quinone side of the RC, electron transfer has relied on the use of a soluble mediator between the RC and an electrode, in part because the probability of QB quinone reduction is much greater than that of direct electron transfer through the large cytoplasmic domain of the H subunit, presenting a ~ 25 Å barrier. A series of C-terminal truncations of the H subunit were created to expose the quinone region of the RC L and M proteins, and all truncated RC H mutants assembled in vivo. The 45M mutant was designed to contain only the N-terminal 45 amino acid residues of the H subunit including the membrane-spanning α-helix; the mutant RC was stable when purified using the detergent N-dodecyl-β-D-maltoside, contained a near-native ratio of bacteriochlorophylls to bacteriopheophytins, and showed a charge-separated state of [Formula: see text]. The 45M-M229 mutant RC had a Cys residue introduced in the vicinity of the QA quinone on the newly exposed protein surface for electrode attachment, decreasing the distance between the quinone and electrode to ~ 12 Å. Steady-state photocurrents of up to around 200 nA/cm² were generated in the presence of 20 mM hydroquinone as the electron donor to the RC. This novel configuration yielded photocurrents orders of magnitude greater than previous reports of electron transfer from the quinone region of RCs bound in this orientation to an electrode.
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