Development and Dynamics of the Photosynthetic Apparatus in Rhodobacter sphaeroides

Geoffrey Chanb and Robert A. Niedermana
aDepartment of Molecular Biology and Biochemistry, Rutgers Energy Institute; bRutgers University/New Brunswick, Piscataway, NJ 08854-8082, USA

Owing to the unique combination of metabolic capabilities and accessible molecular genetics with an intracytoplasmic membrane (ICM) amenable to an unparalleled variety of biochemical, spectroscopic and ultrastructural probes, the purple photo-heterotrophic bacterium Rhodobacter sphaeroides provides an ideal model for exam-ining development and dynamics of energy-transducing membranes. Since very little is known about assembly factors involved in ICM insertion of components of the light-harvesting (LH) and reaction center (RC) complexes, we subjected cells undergoing induction of ICM formation at low oxygen tension to carbonyl-cyanide m-chloro-phenyl-hydrazone (CCCP), [1] in order to block the electrochemical proton gradient necessary for their membrane intregration.

Fig. 1. Effects of CCCP on development of the ICM in the Rba. sphaeroides NF57. In addition to the major chromatophore band, this LH2 only strain, as well as for the wild type, form an upper pigmented band arising from ICM growth initiation when French-press extracts are subjected to rate–zone centrifugation on sucrose density gradients.

A LC-MS/MS analysis of ICM growth iniation sites (Fig. 1) showed ~2-fold elevations in spectral counts for the SecA translocation ATPase, the preprotein translocase SecY, the SecD and SecF insertion components, and chaperonins DnaJ and DnaK, which function early in the assembly process. These factors apparently accumulated with their nascent polypeptides, as putative assembly intermediates in a functionally arrested state. It is also shown in Fig. 1 that CCCP causes an  accumulation of the majority of the photosynthetic pigment in sites of membrane growth initiation. This further establishes that blocking of the electrochemical proton gradient arrests membrane assembly at an early stage insofar as the ICM maturation proces is largely arrested.

Alterations in membrane dynamics as the developing membrane becomes filled with LH2-rings were assessed from fluorescence induction/relaxation kinetics, which showed a slowing in the RC electron transfer turnover rate [1] thought to mainly reflect alterations in donor side electron transfer. This was attributed to an increased distance for electron flow in cytochrome c2 between the RC and cytochrome bc1 complexes, as suggested in current structural models.

Reference

[1] Niederman, R.A. Biochim. Biophys. Acta 2016, 1857, 232–246.