Norbert A. Dencher

Structural biology. Proton-powered turbine of a plant motor.

ATP synthases are enzymes that can work in two directions to catalyse either the synthesis or breakdown of ATP, and they constitute the smallest rotary motors in biology. The flow of protons propels the rotation of a membrane-spanning complex of identical protein subunits, the number of which determines the efficiency of energy conversion. This proton-powered turbine is predicted to consist of 12 subunits, based on data for Escherichia coli. The yeast mitochondrial enzyme, however, has only 10 subunits. We have imaged the ATP synthase from leaf chloroplasts by using atomic force microscopy and, surprisingly, find that its turbine has 14 subunits, arranged in a cylindrical ring.

Cardiolipin: a proton trap for oxidative phosphorylation

The role of specific lipid structures in biological membranes has been elusive. There are hundreds of them in nature. Why has nature made them? How do they aid in the functioning of membrane proteins? Genetics with its ‘knock out’ organisms declares that functions persist in the absence of any particular lipid. Nonetheless some lipids, such as cardiolipin (CL), are associated with particular functions in the cell. It may merely expand the variety of culture conditions (pH, temperature, etc.) under which the wild‐type organism survives. This article explores a unique role of CL as a proton trap within membranes that conduct oxidative phosphorylation and therefore the synthesis of ATP. CLs pK2 (above 8.0) provides a role for it as a headgroup proton trap for oxidative phosphorylation. It suggests why CL is found in membranes that pump protons. The high pK2 also indicates that the headgroup has but one negative charge in the neutral pH range. Data on the binding of CL to all of the oxidative phosphorylation proteins suggest that the CL may aggregate the oxidative phosphorylation proteins into a patch while it restricts pumped protons within its headgroup domain – supplying protons to the ATP synthase with minimal changes in the bulk phase pH.

Architecture of Active Mammalian Respiratory Chain Supercomplexes

In the inner mitochondrial membrane, the respiratory chain complexes generate an electrochemical proton gradient, which is utilized to synthesize most of the cellular ATP. According to an increasing number of biochemical studies, these complexes are assembled into supercomplexes. However, little is known about the architecture of the proposed multicomplex assemblies. Here, we report the electron microscopic characterization of the two respiratory chain supercomplexes I1III2 and I1III2IV1 in bovine heart mitochondria, which are also two major supercomplexes in human mitochondria. After purification and demonstration of enzymatic activity, their structures in projection were determined by single particle image analysis. A difference map between the supercomplexes I1III2 and I1III2IV1 closely fits the x-ray structure of monocomplex IV and shows its location in the assembly. By comparing different views of supercomplex I1III2IV1, the location and mutual arrangement of complex I and the complex III dimer are discussed. Detailed knowledge of the architecture of the active supercomplexes is a prerequisite for a deeper understanding of energy conversion by mitochondria in mammals.

Proton-powered turbine of a plant motor

ATP synthases are enzymes that can work in two directions to catalyse either the synthesis or breakdown of ATP, and they constitute the smallest rotary motors in biology. The flow of protons propels the rotation of a membrane-spanning complex of identical protein subunits, the number of which determines the efficiency of energy conversion. This proton-powered turbine is predicted to consist of 12 subunits, based on data for Escherichia coli. The yeast mitochondrial enzyme, however, has only 10 subunits. We have imaged the ATP synthase from leaf chloroplasts by using atomic force microscopy and, surprisingly, find that its turbine has 14 subunits, arranged in a cylindrical ring.

Time-resolved X-ray diffraction study of structural changes associated with the photocycle of bacteriorhodopsin.

The time course of structural changes accompanying the transition from the M412 intermediate to the BR568 ground state in the photocycle of bacteriorhodopsin (BR) from Halobacterium halobium was studied at room temperature with a time resolution of 15 ms using synchrotron radiation X‐ray diffraction. The M412 decay rate was slowed down by employing mutated BR Asp96Asn in purple membranes at two different pH‐values. The observed light‐induced intensity changes of in‐plane X‐ray reflections were fully reversible. For the mutated BR at neutral pH the kinetics of the structural alterations (tau 1/2 = 125 ms) were very similar to those of the optical changes characterizing the M412 decay, whereas at pH 9.6 the structural relaxation (tau 1/2 = 3 s) slightly lagged behind the absorbance changes at 410 nm. The overall X‐ray intensity change between the M412 intermediate and the ground state was about 9% for the different samples investigated and is associated with electron density changes close to helix G, B and E. Similar changes (tau 1/2 = 1.3–3.6 s), which also confirm earlier neutron scattering results on the BR568 and M412 intermediates trapped at ‐180 degrees C, were observed with wild type BR retarded by 2 M guanidine hydrochloride (pH 9.4). The results unequivocally prove that the tertiary structure of BR changes during the photocycle.

Two photosystems controlling behavioural responses of Halobacterium halobium.

Halobacterium halobium contains a retinal–protein complex, bacteriorhodopsin, which is the only protein of the so-called purple membrane forming distinct patches within the surface membrane1,2. Bacteriorhodopsin acts as a photoreceptor molecule and is chemically similar to the visual pigment rhodopsin1,3. On illumination bacteriorhodopsin undergoes a fast cyclic photoreaction with at least four intermediates occurring after microseconds and milliseconds4–6. Various experiments indicate that bacteriorhodopsin functions as a light-driven proton pump which builds up a proton gradient across the cell membrane and is used for ATP synthesis7,8. Thus its primary function, different from that of rhodopsin in the eye, seems to be energy transformation7. Moreover, H. halobium shows light dependent motor responses, and so we supposed that bacteriorhodopsin also has a sensory function. To test this possibility we obtained action spectra for the light-induced behavioural responses from H. halobium. We found two photosystems, one of which shows that bacteriorhodopsin is involved in the light-controlled motor response.

A natural CD label to probe the structure of the purple membrane from Halobacterium halobium by means of exciton coupling effects.

Coupling between retinal chromophores on adjacent bacteriorhodopsin molecules in the hexagonal surface lattice of the purple membrane from Halobacterium halobium R1 leads to exciton circular dichroism (CD) spectra in the 567 nm absorption band. Uncoupling by solubilization of bacteriorhodopsin in Triton X-100 results in a loss of this couplet. In dimethylsulfoxide (DMSO)/water mixtures, both exciton peaks disappear and a positive CD band develops at 460 nm. The change is reversible. It is suggested that DMSO increases the protein mobility in the membrane. Bleaching causes the concerted disappearance of both exciton peaks around 567 nm, and the appearance of optical activity in the 412 nm band. Because of its strong dependence on geometry, this CD effect appears to be a sensitive probe to study changes in protein mobility and in protein-protein interactions.

Water molecules and exchangeable hydrogen ions at the active centre of bacteriorhodopsin localized by neutron diffraction : elements of the proton pathway ?

Neutron diffraction is used to localize water molecules and/or exchangeable hydrogen ions in the purple membrane by H2O/2H2O exchange experiments at different values of relative humidity. At 100% relative humidity, differences in the hydration between protein and lipid areas are observed, accounting for an excess amount of about 100 molecules of water in the lipid domains per unit cell. A pronounced isotope effect was observed, reproducibly showing an increase in the lamellar spacing from 60 A in 2H2O to 68 A in H2O. At 15% relative humidity, the positions of exchangeable protons became visible. A dominant difference density peak corresponding to 11 +/- 2 exchangeable protons was detected in the central part of the projected structure of bacteriorhodopsin at the Schiffs base end of the chromophore. A difference density map obtained from data on purple membrane films at 15% relative humidity in 2H2O, and the same sample after complete drying in vacuum, revealed that about eight of these protons belong to four water molecules. This is direct evidence for tightly bound water molecules close to the chromophore binding site of bacteriorhodopsin, which could participate in the active steps of H+ translocation as well as in the proton pathway across this membrane protein.

Supramolecular Organization of Cytochrome c Oxidase- and Alternative Oxidase-dependent Respiratory Chains in the Filamentous Fungus Podospora anserina

To elucidate the molecular basis of the link between respiration and longevity, we have studied the organization of the respiratory chain of a wild-type strain and of two long-lived mutants of the filamentous fungus Podospora anserina. This established aging model is able to respire by either the standard or the alternative pathway. In the latter pathway, electrons are directly transferred from ubiquinol to the alternative oxidase and thus bypass complexes III and IV. We show that the cytochrome c oxidase pathway is organized according to the mammalian “respirasome” model (Schägger, H., and Pfeiffer, K. (2000) EMBO J. 19, 1777–1783). In contrast, the alternative pathway is composed of distinct supercomplexes of complexes I and III (i.e. I2 and I2III2), which have not been described so far. Enzymatic analysis reveals distinct functional properties of complexes I and III belonging to either cytochrome c oxidase- or alternative oxidase-dependent pathways. By a gentle colorless-native PAGE, almost all of the ATP synthases from mitochondria respiring by either pathway were preserved in the dimeric state. Our data are of significance for the understanding of both respiratory pathways as well as lifespan control and aging.

Picosecond molecular motions in bacteriorhodopsin from neutron scattering

The characteristics of internal molecular motions of bacteriorhodopsin in the purple membrane have been studied by quasielastic incoherent neutron scattering. Because of the quasihomogeneous distribution of hydrogen atoms in biological molecules, this technique enables one to study a wide variety of intramolecular motions, especially those occurring in the picosecond to nanosecond time scale. We performed measurements at different energy resolutions with samples at various hydration levels within a temperature range of 10-300 K. The analysis of the data revealed a dynamical transition at temperatures Td between 180 K and 220 K for all motions resolved at time scales ranging from 0.1 to a few hundred picoseconds. Whereas below Td the motions are purely vibrational, they are predominantly diffusive above Td, characterized by an enormously broad distribution of correlation times. The variation of the hydration level, on the other hand, mainly affects motions slower than a few picoseconds.