Aberrant Assembly Complexes of the Reaction Center Light-harvesting 1 PufX (RC-LH1-PufX) Core Complex of Rhodobacter sphaeroides Imaged by Atomic Force Microscopy

John D Olsen,Peter G Adams,Philip J Jackson,Mark J Dickman,Pu Qian,C Neil Hunter

doi:10.1074/jbc.m114.596585

Abstract

In the purple phototrophic bacterium Rhodobacter sphaeroides, many protein complexes congregate within the membrane to form operational photosynthetic units consisting of arrays of light-harvesting LH2 complexes and monomeric and dimeric reaction center (RC)-light-harvesting 1 (LH1)-PufX "core" complexes. Each half of a dimer complex consists of a RC surrounded by 14 LH1 αβ subunits, with two bacteriochlorophylls (Bchls) sandwiched between each αβ pair of transmembrane helices. We used atomic force microscopy (AFM) to investigate the assembly of single molecules of the RC-LH1-PufX complex using membranes prepared from LH2-minus mutants. When the RC and PufX components were also absent, AFM revealed a series of LH1 variants where the repeating α(1)β(1)(Bchl)2 units had formed rings of variable size, ellipses, and spirals and also arcs that could be assembly products. The spiral complexes occur when the LH1 ring has failed to close, and short arcs are suggestive of prematurely terminated LH1 complex assembly. In the absence of RCs, we occasionally observed captive proteins enclosed by the LH1 ring. When production of LH1 units was restricted by lowering the relative levels of the cognate pufBA transcript, we imaged a mixture of complete RC-LH1 core complexes, empty LH1 rings, and isolated RCs, leading us to conclude that once a RC associates with the first α1β1(Bchl)2 subunit, cooperative associations between subsequent subunits and the RC tend to drive LH1 ring assembly to completion.

Highlights

Photosynthesis relies upon connected pigment protein complexes to capture, transfer, and utilize light energy
Aberrant Assembly of the light-harvesting 1 (LH1) Ring in the Absence of PufX and reaction center (RC) Complexes: the Formation of Variable Ring Sizes and Morphologies and the Presence of Short Arcs and Linear LH1 Assemblies—Peak force tapping atomic force microscopy (AFM) was used to image a representative range of membrane patches from the LH1-only mutant DD13(pRKEK1) at a resolution high enough to visualize the size and shape of the LH1 ring (Fig. 1)
We note the lack of any protein within the majority of the LH1 complexes imaged, which indicates that the RC H-subunit does not appear to associate with the LH1 complex in the absence of the RC L and M subunits (Fig. 1, A and B)

Summary

Background

Photosynthesis relies upon connected pigment protein complexes to capture, transfer, and utilize light energy. When production of LH1 units was restricted by lowering the relative levels of the cognate pufBA transcript, we imaged a mixture of complete RC-LH1 core complexes, empty LH1 rings, and isolated RCs, leading us to conclude that once a RC associates with the first ␣1␤1(Bchl) subunit, cooperative associations between subsequent subunits and the RC tend to drive LH1 ring assembly to completion. Reducing the levels of ␣1␤1(Bchl) subunits resulted in a subpopulation of isolated RCs that had no evidence of even a partial LH1 complex associated with them, whereas the rest of the RC-LH1 complexes had a complete ring These single molecule observations reveal complexities of photosystem assembly that would not be detected in ensembles by bulk electrophoretic or spectroscopic techniques. We propose a mechanism for formation of ␣1␤1(Bchl) subunits and a sequence for their assembly round the RC

EXPERIMENTAL PROCEDURES

RESULTS

DISCUSSION