دانلود رایگان مقاله مکانیسم ubiquinone واسطه نسل نوری توسط مرکز واکنش بر اساس فوتوکاتد

ABSTRACT

Upon photoexcitation, the reaction center (RC) pigment-proteins that facilitate natural photosynthesis achieve a metastable separation of electrical charge among the embedded cofactors. Because of the high quantum efficiency of this process, there is a growing interest in their incorporation into biohybrid materials for solar energy conversion, bioelectronics and biosensing. Multiple bioelectrochemical studies have shown that reaction centers from various photosynthetic organisms can be interfaced with diverse electrode materials for the generation of photocurrents, but many mechanistic aspects of native protein functionality in a non-native environment is unknown. In vivo, RC's catalyse ubiquinone-10 reduction, protonation and exchange with other lipid phase ubiquinone-10s via protein-controlled spatial orientation and protein rearrangement. In contrast, the mechanism of ubiquinone-0 reduction, used to facilitate fast RC turnover in an aqueous photoelectrochemical cell (PEC), may not proceed via the same pathway as the native cofactor. In this report we show truncation of the native isoprene tail results in larger RC turnover rates in a PEC despite the removal of the tail's purported role of ubiquinone headgroup orientation and binding. Through the use of reaction centers with single or double mutations, we also show the extent to which two-electron/two-proton ubiquinone chemistry that operates in vivo also underpins the ubiquinone-0 reduction by surface-adsorbed RCs in a PEC. This reveals that only the ubiquinone headgroup is critical to the fast turnover of the RC in a PEC and provides insight into design principles for the development of new biophotovoltaic cells and biosensors.

5. Materials and methods

5.1. Experimental materials A strain of Rba. sphaeroides expressing His-tagged WT RCs was constructed as described previously [10]. To construct the EL212W RC, residue Glu 212 of the L-polypeptide was changed to Trp, and to construct he EL212A/DL213A RC residues Glu 212 and Asp 213 of this polypeptide were both changed to Ala. All mutations were introduced using the QuikChange method (Stratagene), were restricted to the target codons and were confirmed by DNA sequencing. For X-ray crystallography, mutations were introduced into plasmid pUCXB-1 which is a derivative of pUC19 containing a 1841 bp XbaI–BamHI fragment encompassing the genes for the RC L and M polypeptides [35]. XbaI/ BamHI restriction fragments containing the mutations were shuttled into expression vector pRKEH10D [58]. For all other work, mutations were introduced into plasmid pv102, which was plasmid pUCXB-1 modified with a sequence that placed ten His residues at the Cterminus of the RC M-polypeptide [10]. XbaI/BamHI restriction fragments containing the mutations were then shuttled into expression vector pv4, which is a derivative of broad-host-range vector pRK415 containing a 6.2 kb EcoRI–HindIII fragment encoding pufQLM; this vector allows expression of the RC L and M polypeptides in the absence of the α and β polypeptides of the LH1 antenna complex or the PufX polypeptide. In both cases the expression vector was introduced into Rba. sphaeroides deletion strain DD13 by conjugative transfer as described previously [59]. Both procedures yielded transconjugant strains that had mutant RCs but lacked both types of Rba. sphaeroides lightharvesting complex, and these were grown under dark/semi-aerobic conditions at 34 °C and 180 rpm in M22+ medium as described elsewhere [58]. Bacterial cells were harvested by centrifugation.