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

Abstract

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 Q 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 Q 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 Q 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/cm 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.

Highly Sensitive Method to Isolate Photocurrent Signals from Large Background Redox Currents on Protein‐Modified Electrodes

“…A stable photocurrent phase response of about 0°was measured from 0.1 to 0.6 V for the conditions with 10 mM HQ and the LED on (Figure 5a, black), which corresponded to the same potential range as the case where non-zero photocurrent was detected (Figure 5a, green). In this RC, Q B is absent [11] and the response therefore validates the finding that electrons are transferred from Q A À directly to the electrode ( Figure 1C) -the electrons cannot proceed from Q A À to Q B due to the absence of the Q B acceptor, and the RC response on the electrode is faster than the back reaction from Q A À to P + . After chargeseparation resulting in the P + Q A À state, the two most-probable electron reactions from Q A À are the back reaction from Q A À to P + with a time constant of 100 ms [7] or the forward reaction towards the electrode.…”

“…[11] When the RC was bound to a gold electrode, CVs showed attenuated HQ redox peaks (Figure 2, black) as compared to bare gold ( Figure 2, red), similar to the MCHmodified electrode surface. [11] When the RC was bound to a gold electrode, CVs showed attenuated HQ redox peaks (Figure 2, black) as compared to bare gold ( Figure 2, red), similar to the MCHmodified electrode surface.…”

“…[11] The RC was purified using Ni 2 + -NTA affinity chromatography as previously described, [11] and after elution from the Ni 2 + -NTA the RCs were exchanged into PBS (1.8 mM KH 2 PO 4 , 10 mM Na 2 HPO 4 , 2.7 mM KCl, 137 mM NaCl) pH 7.2 containing 0.02 % N-dodecyl β-D-maltoside (DDM) detergent using a Centricon centrifugation device. The key difference between the 45M-M229 and the wild type RC is that the former has been genetically modified by removing the H protein cytoplasmic domain, and the result of these changes are that electron transfer does not proceed beyond Q A to Q B , as Q B is absent after purification, and an introduced Cys residue can be used to link the Q-side of the RC to an electrode.…”

“…HQ was used as the sacrificial electron donor in solution to complete the electrical circuit because it reduces the P + charge-separated state of the RC ( Figure 1C). [11] A sacrificial electron donor hydroquinone (HQ) reduces the P + state to restart the cycle; however, in exposed regions of the electrode, HQ can undergo redox reactions, contributing to the background current. Under these conditions in PBS pH 7.2 the first electron transfer of HQ is favorable in reducing P + , which has a midpoint potential of approximately 0.5 V. [29] The coverage of the molecular blocking layer on the gold electrode was estimated using cyclic voltammetry (CV) measured in the dark.…”

“…Numerous studies have investigated the binding of photoactive proteins in various orientations, such as the P-side or Q-side to a bare electrode or surface-modified electrodes using self-assembled linkers. [11][12][13][14][15][16][17][18][19][20][21][22][23] Upon illumination and resultant charge separation, electrons are transferred to the protein from the electrode or from the protein to the electrode depending on the orientation of the protein. Photocurrent generation is, therefore, a function of the number of functional and properly-oriented proteins on the electrode surface, and signals obtained from biohybrid solar cells are typically on the order of nanoamps to microamps per cm 2 .…”