ABSTRACT
In higher plants, chloroplast ATP synthase has a unique redox switch on its γ subunit that modulates enzyme activity to limit ATP hydrolysis at night. To understand the molecular details of the redox modulation, we used single-particle cryo-EM to determine the structures of spinach chloroplast ATP synthase in both reduced and oxidized states. The disulfide linkage of the oxidized γ subunit introduces a torsional constraint to stabilize the two ß hairpin structures. Once reduced, free cysteines alleviate this constraint, resulting in a concerted motion of the enzyme complex and a smooth transition between rotary states to facilitate the ATP synthesis. We added an uncompetitive inhibitor, tentoxin, in the reduced sample to limit the flexibility of the enzyme and obtained high-resolution details. Our cryo-EM structures provide mechanistic insight into the redox modulation of the energy regulation activity of chloroplast ATP synthase.
Subject(s)
Chloroplast Proton-Translocating ATPases/chemistry , Chloroplast Proton-Translocating ATPases/metabolism , Spinacia oleracea/enzymology , Biocatalysis , Chloroplast Proton-Translocating ATPases/ultrastructure , Cryoelectron Microscopy , Light , Models, Biological , Models, Molecular , Nucleotides/metabolism , Oxidation-Reduction , Protein Domains , Protein Subunits/chemistry , Statistics as Topic , Structure-Activity RelationshipABSTRACT
F1Fo ATP synthases produce most of the ATP in the cell. F-type ATP synthases have been investigated for more than 50 years, but a full understanding of their molecular mechanisms has become possible only with the recent structures of complete, functionally competent complexes determined by electron cryo-microscopy (cryo-EM). High-resolution cryo-EM structures offer a wealth of unexpected new insights. The catalytic F1 head rotates with the central γ-subunit for the first part of each ATP-generating power stroke. Joint rotation is enabled by subunit δ/OSCP acting as a flexible hinge between F1 and the peripheral stalk. Subunit a conducts protons to and from the c-ring rotor through two conserved aqueous channels. The channels are separated by â¼6 Å in the hydrophobic core of Fo, resulting in a strong local field that generates torque to drive rotary catalysis in F1. The structure of the chloroplast F1Fo complex explains how ATPase activity is turned off at night by a redox switch. Structures of mitochondrial ATP synthase dimers indicate how they shape the inner membrane cristae. The new cryo-EM structures complete our picture of the ATP synthases and reveal the unique mechanism by which they transform an electrochemical membrane potential into biologically useful chemical energy.
Subject(s)
Adenosine Triphosphate/metabolism , Proton-Translocating ATPases/metabolism , Adenosine Triphosphate/biosynthesis , Animals , Bacteria/enzymology , Bacteria/metabolism , Chloroplast Proton-Translocating ATPases/chemistry , Chloroplast Proton-Translocating ATPases/metabolism , Chloroplast Proton-Translocating ATPases/ultrastructure , Chloroplasts/enzymology , Cryoelectron Microscopy , Eukaryota/enzymology , Eukaryota/metabolism , Humans , Mitochondria/enzymology , Mitochondrial Proton-Translocating ATPases/chemistry , Mitochondrial Proton-Translocating ATPases/metabolism , Mitochondrial Proton-Translocating ATPases/ultrastructure , Protein Conformation , Protein Subunits , Proton-Translocating ATPases/chemistry , Proton-Translocating ATPases/ultrastructureABSTRACT
Envelope-free chloroplasts were imaged in situ by contact and tapping mode scanning force microscopy at a lateral resolution of 3-5 nm and vertical resolution of approximately 0.3 nm. The images of the intact thylakoids revealed detailed structural features of their surface, including individual protein complexes over stroma, grana margin and grana-end membrane domains. Structural and immunogold-assisted assignment of two of these complexes, photosystem I (PS I) and ATP synthase, allowed direct determination of their surface density, which, for both, was found to be highest in grana margins. Surface rearrangements and pigment- protein complex redistribution associated with salt-induced membrane unstacking were followed on native, hydrated specimens. Unstacking was accompanied by a substantial increase in grana diameter and, eventually, led to their merging with the stroma lamellae. Concomitantly, PS IIalpha effective antenna size decreased by 21% and the mean size of membrane particles increased substantially, consistent with attachment of mobile light-harvesting complex II to PS I. The ability to image intact photosynthetic membranes at molecular resolution, as demonstrated here, opens up new vistas to investigate thylakoid structure and function.
Subject(s)
Intracellular Membranes/ultrastructure , Microscopy, Atomic Force , Photosynthetic Reaction Center Complex Proteins/ultrastructure , Thylakoids/ultrastructure , Chloroplast Proton-Translocating ATPases/ultrastructure , Imaging, Three-Dimensional , Immunohistochemistry , Lactuca , Microscopy, Electron , Particle Size , Photosystem I Protein ComplexABSTRACT
The atomic force microscope (AFM) is an exquisitely delicate probe measuring the height of a specimen at discrete sampling points in a fixed two-dimensional (2D) raster. The resulting topograph is a 2D digital image, with each pixel representing a distinct height measurement. The height of an object is determined as the average of the maximum heights measured above the supporting surface. We show that such object heights derived from a variety of organic samples depend critically on the sampling or pixel size of the 2D raster. It is concluded that to obtain accurate specimen heights, the pixel size must be small enough to resolve submolecular structures and thus ensure representative sampling of the height variation on the surface.
