Identification of intramembrane hydrogen bonding between 131 keto group of bacteriochlorophyll and serine residue α27 in the LH2 light-harvesting complex

Abstract

Intramembrane hydrogen bonding and its effect on the structural integrity of purple bacterial light-harvesting complex 2, LH2, have been assessed in the native membrane environment. A novel hydrogen bond has been identified by Raman resonance spectroscopy between a serine residue of the membrane-spanning region of LH2 alpha-subunit, and the C-13(1) keto carbonyl of bacteriochlorophyll (BChl) B850 bound to the beta-subunit. Replacement of the serine by alanine disrupts this strong hydrogen bond, but this neither alters the strongly red-shifted absorption nor the structural arrangement of the BChls, as judged from circular dichroism. It also decreases only slightly the thermal stability of the mutated LH2 in the native membrane environment. The possibility is discussed that weak H-bonding between the C-13(1) keto carbonyl and a methyl hydrogen of the alanine replacing serine(-4) or the imidazole group of the nearby histidine maintains structural integrity in this very stable bacterial light-harvesting complex. A more widespread occurrence of H-bonding to C-13(1) not only in BChl, but also in chlorophyll proteins, is indicated by a theoretical analysis of chlorophyll/polypeptide contacts at <3.5 A in the high-resolution structure of Photosystem I. Nearly half of the 96 chlorophylls have aa residues suitable as hydrogen bond donors to their keto groups.

Hydrogen Bonding in a Model Bacteriochlorophyll-binding Site Drives Assembly of Light Harvesting Complex

“…In Vivo Assembly of Model BChl Proteins-The assembly of the complexes is monitored by absorption spectroscopy, because significant spectral alterations accompany the BChlBChl and BChl-polypeptide association to the native (␣␤) 9 or similar complexes. The near infrared absorption spectra of purified membranes of LH2-like complexes in which only one subunit has been replaced by the model TMH with the Ala-Leu stretch are similar to the spectra of wt LH2 complexes ( TMH AL , typical for the B800 pigments, and to 849 nm in wt and 848 -53 nm in TMH AL , typical for the B850 pigments in LH2 complexes.…”

“…Considering that the optimal growth temperature is ϳ34°C for R. sphaeroides, it is little surprising that ␣AL is found in the membrane at considerable lower levels than wt LH2 (32). In LH2 with ␣AL S Ϫ4-␤wt, which is identical to ␣AL-␤wt except for the exchange of alanine by serine at position Ϫ4, the T m is significantly shifted to higher temperatures (T m ϭ 55°C) approaching the T m of wt LH2 (T m ϭ 69°C) (9). Interestingly, in LH2 with ␣AL S Ϫ4-␤AL, the T m is ϳ42°C, which is considerably higher than the T m of ␣AL-␤wt (Fig.…”

“…Thermal Denaturation of LH2-Thermal denaturation of membranes containing LH2 from R. sphaeroides DD13 was carried out as described (9). In essence, purified membranes, wt LH2 and LH2 ␣S(Ϫ4)A were adjusted to an A 850 nm ϭ ϳ3 cm Ϫ1 in Tris (10 mM, pH 8) EDTA (1 mM) and measured in a 1-mm cylindrical quartz cuvette.…”

“…It has also been suggested that hydrogen bonding to the acetyl group contributes to the tuning of the redox energies of P870 in bacterial reaction centers (5), but this is still under some dispute (6). Hydrogen bonding to the C13 1 keto carbonyl at the isocyclic ring, which is common to all chlorophylls, seems to be widespread (6 -9) but appears to have less or no effect on the electronic properties of (B)Chl (6,9,10). In the crystal structure of photosystem I reaction center (2), which comprises more than 10 TM subunits and contains nearly 100 Chl-binding pockets, the majority of the polar groups of Chls, in particular the C13 1 keto group, are likely to be hydrogen-bonded by the polypeptide residues in close vicinity (9,11,12).…”

“…Hydrogen bonding to the C13 1 keto carbonyl at the isocyclic ring, which is common to all chlorophylls, seems to be widespread (6 -9) but appears to have less or no effect on the electronic properties of (B)Chl (6,9,10). In the crystal structure of photosystem I reaction center (2), which comprises more than 10 TM subunits and contains nearly 100 Chl-binding pockets, the majority of the polar groups of Chls, in particular the C13 1 keto group, are likely to be hydrogen-bonded by the polypeptide residues in close vicinity (9,11,12). It is not exactly understood, however, whether these hydrogen bonds essentially contribute to the structural stability of the membraneembedded (B)Chl proteins.…”