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

Abstract

In this study, the contribution of intramembrane hydrogen bonding at the interface between polypeptide and cofactor is explored in the native lipid environment by use of model bacteriochlorophyll proteins. In the peripheral antenna complex, LH2, large portions of the transmembrane helices, which make up the dimeric bacteriochlorophyll-binding site, are replaced by simplified, alternating alanine-leucine stretches. Replacement of either one of the two helices with the helices containing the model sequence at a time results in the assembly of complexes with nearly native light harvesting properties. In contrast, replacement of both helices results in the loss of antenna complexes from the membrane. The assembly of such doubly modified complexes is restored by a single intramembrane serine residue at position ؊4 relative to the liganding histidine of the ␣-subunit. In situ analysis of the spectral properties in a series of site-directed mutants reveals a critical dependence of the model complex assembly on the side chain of the residue at this position in the helix. A hydrogen bond between the hydroxy group of the serine and the 13 1 keto group of one of the central bacteriochlorophylls of the complexes is identified by Raman spectroscopy in the model antenna complex containing one of the alanine-leucine helices. The additional OH group of the serine residue, which participates in hydrogen bonding, increases the thermal stability of the model complexes in the native membrane. Intramembrane hydrogen bonding is thus shown to be a key factor for the binding of bacteriochlorophyll and assembly of this model cofactor-polypeptide site.

High pressure near infrared study of the mutated light-harvesting complex LH2

“…In spite of the extensive mutations, the spectral properties of these complexes remain unchanged (9,10). However, the mutations induced local packing defects around the BChl-B850 molecules ( Figure 2D) and a significant reduction in thermal stability.…”

Structural Role of (Bacterio)chlorophyll Ligated in the Energetically Unfavorable β-Position

“…This band, which is sensitive to the conformation of the (B)Chl macrocycle, may be used for comparing the intensity of the different bands from the carbonyl stretching modes, thus assessing what happens in this spectral region upon, for example, a mutation. In the RR spectrum of WT LH2, five bands in the carbonyl stretching mode region (1620 -1710 cm Ϫ1 ) are resolved ( have been attributed to the C-3 acetyl groups of the BChl-B850 (27). The stretching mode of the acetyl carbonyl of the BChl-B800 also contributes in this frequency range, but it is very weak in FT-Raman spectra (39).…”

“…Preparation of Intracytoplasmic Membranes-The membranes were prepared from cells grown semi-aerobically in the dark by disruption in a French pressure cell and subsequent centrifugation on a sucrose step gradient (27).…”

“…Instrumentation-In situ absorbance spectroscopy of whole cells in bacterial colonies was performed on a dual fiber model SD2000 spectrophotometer equipped with a reflexion probe (Avantes, Ostfildern, Germany) as described previously (11,27). FT-Raman spectra and CD spectra of purified membranes and isolated LH2s were recorded using a Bruker IFS 66 interferometer coupled to a Bruker FRA 106 Raman module equipped with a continuous Nd:YAG laser and on a Dichrograph (Jasco J-715) as described previously (28).…”

Spectral properties of LH2 exhibit very similar even when heterologously express LH2 with <i>β</i>-subunit fusion protein in <i>Rhodobacter</i> <i>sphaeroides</i>

“…Hydrogen bonding environment largely influences LH2 absorbance [16] and the H-bonding was also revealed to drive the assembly of LH2 in Rb. sphaeroides [17,18]. Similarly, the LH1 αTrp43 was proposed to be located near the putative binding site for BChl molecule [19].…”