The structures of S0 spheroidene in the light-harvesting (LH2) complex and S0 and T1 spheroidene in the reaction center of Rhodobacter sphaeroides 2.4.1 as revealed by Raman spectroscopy

ABSTRACT The photosynthetic complexes of the thermophile Thermochromatium tepidum are of considerable interest in biohybrid solar cell applications because of the ability of thermophilic proteins to tolerate elevated temperatures. Synthetic operons encoding reaction center (RC) and light harvesting 1 (LH1) pigment-protein complexes of T. tepidum were expressed in the mesophile Rhodobacter sphaeroides. The T. tepidum RC (TRC) was assembled and was found to be functional with the addition of menadione to populate the QA pocket. The production of T. tepidum LH1 (TLH1) was increased by selection of a phototrophy-capable mutant after UV irradiation mutagenesis, which yielded a hybrid RC-TLH1 core complex consisting of the R. sphaeroides RC and T. tepidum TLH1, confirmed by the absorbance peak of TLH1 at 915 nm. Affinity chromatography partial purification and subsequent sucrose gradient analysis of the hybrid RC-TLH1 core complex indicated that this core complex assembled as a monomer. Furthermore, the RC-TLH1 hybrid core complex was more tolerant of a temperature of 70°C than the R. sphaeroides RC-LH1 core complexes in both the dimeric and monomeric forms; after 1 h, the hybrid complex retained 58% of the initial starting value, compared to values of 11% and 53% for the R. sphaeroides RC-LH1 dimer and monomer forms, respectively. IMPORTANCE This work is important because it is a new approach to bioengineering of photosynthesis proteins for potential use in biophotovoltaic solar energy capture. The work establishes a proof of principle for future biohybrid solar cell applications.

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Blue shifts in bacteriochlorophyll absorbance correlate with changed hydrogen bonding patterns in light-harvesting 2 mutants of Rhodobacter sphaeroides with alterations at α-Tyr-44 and α-Tyr-45

Biochemical Journal ◽

10.1042/bj2990695 ◽

1994 ◽

Vol 299 (3) ◽

pp. 695-700 ◽

Cited By ~ 85

Author(s):

G J S Fowler ◽

G D Sockalingum ◽

B Robert ◽

C N Hunter

Keyword(s):

Raman Spectroscopy ◽

Hydrogen Bonds ◽

Rhodobacter Sphaeroides ◽

Light Harvesting ◽

Theoretical Calculations ◽

Resonance Raman Spectroscopy ◽

Resonance Raman ◽

Photosynthetic Bacterium ◽

Site Directed Mutagenesis ◽

Aromatic Residues

A combination of Fourier-Transform (FT) resonance Raman spectroscopy and site-directed mutagenesis has been used to examine the function of two highly conserved aromatic residues, alpha-Tyr-44 and alpha-Tyr-45, in the light-harvesting 2 (LH2) complex of the photosynthetic bacterium Rhodobacter sphaeroides. In LH2 complexes, aromatic residues located at positions alpha-44 and alpha-45 are thought to be located near the putative binding site for bacteriochlorophyll, and alterations at these positions are known to produce blue shifts in bacteriochlorophyll absorbance. In the present work, mutant LH2 complexes carrying the alterations alpha-Tyr-44-->Phe, alpha-Tyr-45-->Phe and alpha-Tyr-44,-45-->Phe,Leu were examined. FT resonance Raman spectroscopy of the resulting complexes shows the breakage of a hydrogen bond to the 2-acetyl carbonyl group of one of the B850 bacteriochlorophylls in the LH2 complex; in the double mutant, breakage of a second bond is probable. These results suggest that one of these hydrogen bonds is to alpha-Tyr-44, placing this residue in close proximity to ring I of one of the B850 bacteriochlorophyll a pigments. The breakage of one, then two, 2-acetyl carbonyl hydrogen bonds correlates well with the shift in the absorbance of the B850 pigments of 11 nm then 26 nm at 77 K. Thus a consistency between literature theoretical calculations and the observations from both absorption and FT resonance Raman spectroscopy is demonstrated.

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