Reconstitution of the heliobacterial photochemical reaction center and cytochrome c553 into a proteoliposome system

• Published in: Photosynthesis research Vol. 143; no. 3; pp. 241 - 250
• Main Authors: Johnson, William A., Redding, Kevin E.
• Format: Journal Article
• Language: English
• Published: Dordrecht Springer Netherlands 01-03-2020; Springer Nature B.V
• Subjects: Binding sites; Biochemistry; Biomedical and Life Sciences; Cytochrome; Diglycerides; Life Sciences; Lipids; Liposomes; Mimicry; Original Article; Phospholipid composition; Phospholipids; Plant Genetics and Genomics; Plant Physiology; Plant Sciences; Recombination; Surface charge; Cytochrome; Heliobacteria; Reconstitution; Reaction center; Proteoliposomes
• ISSN: 0166-8595; 1573-5079
• DOI: 10.1007/s11120-019-00695-w

The heliobacterial reaction center (HbRC) is the simplest known photochemical reaction center, in terms of its polypeptide composition. In the heliobacterial cells, its electron donor is a cytochrome (cyt)
c
553
attached to the membrane via a covalent linkage with a diacylglycerol. We have reconstituted purified HbRC into liposomes mimicking the phospholipid composition of heliobacterial membranes. We also incorporated a lipid with a headgroup containing Ni(II):nitrilotriacetate (NTA) to provide a binding site for the soluble version of the heliobacterial cyt
c
553
in which the N-terminal membrane attachment site is replaced by a hexahistidine tag. The HbRC was inserted into the liposomes with the donor side preferentially exposed to the exterior; this bias increased to nearly 100% with higher concentrations (≥ 10 mol%) of the Ni(II)-NTA lipid in the membrane, and is most likely due to the net negative charge of the surface of the membrane. The HbRC in proteoliposomes without the Ni(II)-NTA lipid exhibited normal charge separation and subsequent charge recombination of the P
800
+
F
X
−
state in 15 ms; however, the oxidized primary donor (P
800
+
) was not significantly reduced by added H
6
-cyt
c
553
. In contrast, with proteoliposomes containing the Ni(II)-NTA lipid, addition of H
6
-cyt
c
553
resulted in a new kinetic component resulting from fast reduction (2–5 ms) of P
800
+
by H
6
-cyt
c
553
. The contribution of this kinetic component varied with the concentration of added H
6
-cyt
c
553
and could represent 80% or more of the total P
800
+
decay. Thus, the HbRC and its interaction with its native electron donor have been reconstituted into an artificial membrane system.