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

Abstract

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.

Bacteriochlorin‐protein interactions in native B800‐B850, B800 deficient and B800‐Bchlap‐reconstituted complexes from Rhodopseudomonas acidophila, strain 10050

“…From these studies, for example, it has been shown that each H-bond breakage between the apoproteins and the acetyl carbonyl oxygens of the B850 molecules correlates with a 10 nm blue shift of the absorption maximum [9]. A similar e¡ect also occurs with the B800 molecules.…”

“…Here, we have compared the Bchla-protein interactions in native and Bchla-reconstituted complexes by resonance Raman spectroscopy. The Raman spectra of LH2 complexes have previously been shown to be very sensitive to the environment of the Bchla molecules [6,9,10]. This allowed us to use this technique to assess whether or not the Bchla molecules are correctly bound within the reconstituted complex.…”

Knowledge based structure prediction of the light-harvesting complex II of rhodospirillum molischianum

“…As mentioned above, the α-Trp-45 is hydrogen-bonded to the 2-acetyl group of BChla. However, there is no direct information about which of the two B850 BChla's is involved in such a hydrogen bond [FSRH94]. Based on the optimized tertiary structure for both α and β subunits,we found that it is more feasible spatially to form an inter-unit hydrogen bond, i.…”

“…rubrum on the cytoplasmic side of the membrane by partial hydrolysis with proteolytic enzymes [BWS + 84], both α-and β-polypeptides should be oriented with their N-terminals towards the cytoplasm; (2) strong circular dichroism (CD) signals suggest exciton interactions between pairs of BChla molecules [Zub93]; (3) linear dichroism data indicate that the B850 BChla's are approximately perpendicular to the membrane and the B800 BChla is parallel to the membrane [KvGH + 84]; (4) Fourier-transform Resonance Raman spectroscopy and site-directed mutagenesis [FSRH94] of a related lightharvesting complex indicate that another highly conserved residue, α-Trp-45, is hydrogen-bonded to the 2-acetyl group of BChla. All these observations impose constraints on the structure.…”

Tansley Review No. 109.

“…850 nm ; hence the alternative names for LH1 and LH2 of B875 and B800-850 respectively. These antenna complexes were also subjected to extensive spectroscopic analysis and genetic manipulation by site-directed mutagenesis (Youvan et al, 1985 ;Burgess et al, 1988 ;Bylina et al, 1988 ;Coleman & Youvan, 1990 ;Fowler et al, 1992Fowler et al, , 1994Jones et al, 1992a ;Hunter, 1995), as a result of which several structural models were proposed. The development of ideas about the structure and function of these antenna complexes is the subject of two reviews by Hunter and co-workers (Hunter et al, 1989 ;Olsen & Hunter, 1994).…”