The insertion of the non-heme FeB cofactor into nitric oxide reductase from P. denitrificans depends on NorQ and NorD accessory proteins

Bacterial NO reductases (NOR) catalyze the reduction of NO into N₂O, either as a step in denitrification or as a detoxification mechanism. cNOR from Paracoccus (P.) denitrificans is expressed from the norCBQDEF operon, but only the NorB and NorC proteins are found in the purified NOR complex.Here, we established a new purification method for the P. denitrificans cNOR via a His-tag using heterologous expression in E. coli. The His-tagged enzyme is both structurally and functionally very similar to non-tagged cNOR. We were also able to express and purify cNOR from the structural genes norCB only, in absence of the accessory genes norQDEF. The produced protein is a stable NorCB complex containing all hemes and it can bind gaseous ligands (CO) to heme b₃, but it is catalytically inactive. We show that this deficient cNOR lacks the non-heme iron cofactor Fe_B. Mutational analysis of the nor gene cluster revealed that it is the norQ and norD genes that are essential to form functional cNOR. NorQ belongs to the family of MoxR P-loop AAA+ ATPases, which are in general considered to facilitate enzyme activation processes often involving metal insertion. Our data indicates that NorQ and NorD work together in order to facilitate non-heme Fe insertion. This is noteworthy since in many cases Fe cofactor binding occurs spontaneously. We further suggest a model for NorQ/D-facilitated metal insertion into cNOR.     

Energy transduction in photosynthetic bacteria IV. Light-dependent ATPase in photosynthetic membranes from Rhodopseudomonas capsulata

ATPase activity of photosynthetic membrane fragments from the bacterium Rhodopseudomonas capsulata can be stimulated by continuous illumination under conditions of active cyclic electron flow. The activation corresponds to an increase in the maximum velocity of the reaction and does not affect the apparent K_m for ATP (0.11 mM). No stimulation in the light is observed in the presence of classical uncouplers or oxidized 2,6-dichlorophenolindophenol (DCIP), which, per se, stimulate ATPase in the dark. It is demonstrated, however, that oxidized DCIP acts as an uncoupler of bacterial photophosphorylation.

The effect of light is elicited after a few minutes of preillumination, or in a much shorter time if an ADP trapping system is supplied. Activation does not occur if ADP is added during the preillumination (apparent K_m for inhibition by ADP = 1 μM). The effect of ADP is not related to competitive inhibition with ATP, which can be observed at higher concentrations (apparent K_i = 0.26 mM). ADP, however, is not effective if added after some minutes of preillumination.     

Respiration in energy-transducing membranes of the thermophilic cyanobacterium Mastigocladus laminosus: II. Oxidative phosphorylation

Oxidative phosphorylation was measured in isolated energy-transducing membranes of the thermophilic cyanobacterium Mastigocladus laminosus with NADH-mediated electron transport. This dark phosphorylation was similar to photophosphorylation in its sensitivity to uncouplers and energy-transfer inhibitors. However, photophosphorylation was 20- to 50-times more active than oxidative phosphorylation. The $\text{P}\text{O}$ ratio of oxidative phosphorylation was about 0.2. Besides oxidative phosphorylation, adenylate kinase- and ADP-P_i exchange activity were measured in the dark. The ADP-P_i exchange reaction was identified as polynucleotide phosphorylase.     

The role of the Rieske iron-sulfur center as the electron donor to ferricytochrome c2 in Rhodopseudomonas sphaeroides

The Rieske iron-sulfur center in the photosynthetic bacterium Rhodopseudomonas sphaeroides appears to be the direct electron donor to ferricytochrome c₂, reducing the cytochrome on a submillisecond timescale which is slower than the rapid phase of cytochrome oxidation ($\text{t}{}_{\mathrm{<mtext>1</mtext><mtext>2</mtext>}}\text{3–5 μ}\text{s}$). The reduction of the ferricytochrome by the Rieske center is inhibited by 5-n-undecyl-6-hydroxy-4,7-dioxobenzothiazole (UHDBT) but not by antimycin. The slower (1–2 ms) antimycin-sensitive phase of ferricytochrome c₂ reduction, attributed to a specific ubiquinone-10 molecule (Q_z), and the associated carotenoid spectral response to membrane potential formation are also inhibited by UHDBT. Since the light-induced oxidation of the Rieske center is only observed in the presence of antimycin, it seems likely that the reduced form of Q_z (Q_zH₂) reduces the Rieske center in an antimycin-sensitive reaction. From the extent of the UHDBT-sensitive ferricytochrome c₂ reduction we estimate that there are 0.7 Rieske iron-sulfur centers per reaction center.UHDBT shifts the EPR derivative absorption spectrum of the Rieske center from g_y 1.90 to g_y 1.89, and shifts the E_m,7 from 280 to 350 mV. While this latter shift may account for the subsequent failure of the iron-sulfur center to reduce ferricytochrome c₂, it is not clear how this can explain the other effects of the inhibitor, such as the prevention of cytochrome b reduction and the elimination of the uptake of H⁺_II; these may reflect additional sites of action of the inhibitor.     

Energy-dependent masking of substrate binding sites of the lactose permease of Escherichia coli

A method was devised to measure the number of specific substrate binding sites of lactose permease in membrane preparations derived from mechanically disrupted Escherichia coli.The method consists of incubation with radioactive thiodigalactoside (galactosyl β-d-thiogalactoside, TDG) followed by precipitation with 80% saturated (NH₄)₂SO₄ and washing with the same solution.The measurement gave reproducible results, easy to correct for a moderate nonspecific binding, but active transport, when it occurred, resulted in excess counts.The radioactivity bound to the pellet was shown to depend on the presence of intact lac y gene product.Addition of ascorbate and phenazine methosulfate (PMS) stimulated active transport into the membrane vesicles. This could be inhibited by cyanide and by uncoupling agents and under these conditions the number of available binding sites was strongly diminished, while the inhibitors alone did not bring about a similar decrease.The decrease of available substrate binding sites was reversed by removal of oxygen or by washing out the respiratory substrates.The decrease in available binding sites is interpreted as reflecting one of the energy coupling steps which during in vivo active transport prevents the mobile carrier from being available for outflux, but the detailed interpretation of the reported results raises a number of problems connected with the energy cycle of active transport     

The ultrastructure and flexibility of thylakoid membranes in leaves and isolated chloroplasts as revealed by small-angle neutron scattering

We studied the periodicity of the multilamellar membrane system of granal chloroplasts in different isolated plant thylakoid membranes, using different suspension media, as well as on different detached leaves and isolated protoplasts—using small-angle neutron scattering. Freshly isolated thylakoid membranes suspended in isotonic or hypertonic media, containing sorbitol supplemented with cations, displayed Bragg peaks typically between 0.019 and 0.023 Å^− 1, corresponding to spatially and statistically averaged repeat distance values of about 275–330 Å. Similar data obtained earlier led us in previous work to propose an origin from the periodicity of stroma thylakoid membranes. However, detached leaves, of eleven different species, infiltrated with or soaked in D₂O in dim laboratory light or transpired with D₂O prior to measurements, exhibited considerably smaller repeat distances, typically between 210 and 230 Å, ruling out a stromal membrane origin. Similar values were obtained on isolated tobacco and spinach protoplasts. When NaCl was used as osmoticum, the Bragg peaks of isolated thylakoid membranes almost coincided with those in the same batch of leaves and the repeat distances were very close to the electron microscopically determined values in the grana. Although neutron scattering and electron microscopy yield somewhat different values, which is not fully understood, we can conclude that small-angle neutron scattering is a suitable technique to study the periodic organization of granal thylakoid membranes in intact leaves under physiological conditions and with a time resolution of minutes or shorter. We also show here, for the first time on leaves, that the periodicity of thylakoid membranes in situ responds dynamically to moderately strong illumination. This article is part of a Special Issue entitled: Photosynthesis research for sustainability: Keys to produce clean energy.     

Evidence for involvement of the electron transport system at a late step of anaerobic microbial heme synthesis

The penultimate step in heme biosynthesis, the oxidation of protoporphyrinogen to protoporphyrin, can be anaerobically coupled to the reduction of fumarate in extracts of anaerobically-grown Escherichia coli. This coupling is approximately 90% inhibited by 2-heptyl-4-hydroxy quinoline-N-oxide (HQNO), a known inhibitor of the electron transport chain. This observation suggests that the mechanism of the anaerobic oxidation of protoporphyrinogen in E. coli involves a coupling into the anaerobic electron transport system. In contrast, the aerobic oxidation of protoporphyrinogen, which occurs in mammalian and yeast mitochondria, is known to be linked directly to oxygen without the mediation of an electron transport system.