Structural basis for electron transport mechanism of complex I-like photosynthetic NAD(P)H dehydrogenase.

NAD(P)H dehydrogenase-like (NDH) complex NDH-1L of cyanobacteria plays a crucial role in cyclic electron flow (CEF) around photosystem I and respiration processes. NDH-1L couples the electron transport from ferredoxin (Fd) to plastoquinone (PQ) and proton pumping from cytoplasm to the lumen that drives the ATP production. NDH-1L-dependent CEF increases the ATP/NADPH ratio, and is therefore pivotal for oxygenic phototrophs to function under stress. Here we report two structures of NDH-1L from Thermosynechococcus elongatus BP-1, in complex with one Fd and an endogenous PQ, respectively. Our structures represent the complete model of cyanobacterial NDH-1L, revealing the binding manner of NDH-1L with Fd and PQ, as well as the structural elements crucial for proper functioning of the NDH-1L complex. Together, our data provides deep insights into the electron transport from Fd to PQ, and its coupling with proton translocation in NDH-1L.

Selectivity through discriminatory induced fit enables switching of NAD(P)H coenzyme specificity in Old Yellow Enzyme ene-reductases.

Most ene-reductases belong to the Old Yellow Enzyme (OYE) family of flavin-dependent oxidoreductases. OYEs use nicotinamide coenzymes as hydride donors to catalyze the reduction of alkenes that contain an electron-withdrawing group. There have been many investigations of the structures and catalytic mechanisms of OYEs. However, the origin of coenzyme specificity in the OYE family is unknown. Structural NMR and X-ray crystallographic data were used to rationally design variants of two OYEs, pentaerythritol tetranitrate reductase (PETNR) and morphinone reductase (MR), to discover the basis of coenzyme selectivity. PETNR has dual-specificity and reacts with NADH and NADPH; MR accepts only NADH as hydride donor. Variants of a ß-hairpin motif in an active site loop of both these enzymes were studied using stopped-flow spectroscopy. Specific attention was placed on the potential role of arginine residues within the ß-hairpin motif. Mutagenesis demonstrated that Arg130 governs the preference of PETNR for NADPH, and that Arg142 interacts with the coenzyme pyrophosphate group. These observations were used to switch coenzyme specificity in MR by replacing either Glu134 or Leu146 with arginine residues. These variants had increased (~15-fold) affinity for NADH. Mutagenesis enabled MR to accept NADPH as a hydride donor, with E134R MR showing a significant (55-fold) increase in efficiency in the reductive half-reaction, when compared to the essentially unreactive wild-type enzyme. Insight into the question of coenzyme selectivity in OYEs has therefore been addressed through rational redesign. This should enable coenzyme selectivity to be improved and switched in other OYEs.

Structure of the complex I-like molecule NDH of oxygenic photosynthesis.

Cyclic electron flow around photosystem I (PSI) is a mechanism by which photosynthetic organisms balance the levels of ATP and NADPH necessary for efficient photosynthesis1,2. NAD(P)H dehydrogenase-like complex (NDH) is a key component of this pathway in most oxygenic photosynthetic organisms3,4 and is the last large photosynthetic membrane-protein complex for which the structure remains unknown. Related to the respiratory NADH dehydrogenase complex (complex I), NDH transfers electrons originating from PSI to the plastoquinone pool while pumping protons across the thylakoid membrane, thereby increasing the amount of ATP produced per NADP+ molecule reduced4,5. NDH possesses 11 of the 14 core complex I subunits, as well as several oxygenic-photosynthesis-specific (OPS) subunits that are conserved from cyanobacteria to plants3,6. However, the three core complex I subunits that are involved in accepting electrons from NAD(P)H are notably absent in NDH3,5,6, and it is therefore not clear how NDH acquires and transfers electrons to plastoquinone. It is proposed that the OPS subunits-specifically NdhS-enable NDH to accept electrons from its electron donor, ferredoxin3-5,7. Here we report a 3.1 Å structure of the 0.42-MDa NDH complex from the thermophilic cyanobacterium Thermosynechococcus elongatus BP-1, obtained by single-particle cryo-electron microscopy. Our maps reveal the structure and arrangement of the principal OPS subunits in the NDH complex, as well as an unexpected cofactor close to the plastoquinone-binding site in the peripheral arm. The location of the OPS subunits supports a role in electron transfer and defines two potential ferredoxin-binding sites at the apex of the peripheral arm. These results suggest that NDH could possess several electron transfer routes, which would serve to maximize plastoquinone reduction and avoid deleterious off-target chemistry of the semi-plastoquinone radical.

[The ultrastructural localization of NO-synthase NADPH diaphorase in a peripheral nerve and its change in diphtheritic polyneuropathy]. / Ul'trastrukturnaia lokalizatsiia NO-sintaznoi NADPH-diaforazy v perifericheskom nerve i ee izmenenie pri difteriinoi polineiropatii.

Light and electron microscopy was used to study the distribution and changes of NADPH-diaphorase in the cutaneous nerve biopsy specimens in different periods of diphtheritic polyneuropathy (DP). there was a reduction in the reaction rate of the enzyme in Schwann's cells of the destructively changed nerve fibers and an increase in the remyelinated nerve fibers. The enzyme is located on the nuclear and endoplasmic reticulum membranes and ribosomes. It is suggested that there is an association of the synthesis of nitric oxide with the myelin-producing function of Schwann's cells.

Ultrastructural demonstration of the NADPH-diaphorase activity in peritubular myoid cells and fibroblasts of the lamina propria of the mouse seminiferous tubules.

The NOS-related NADPH-diaphorase activity was studied by transmission electron microscopy in the peritubular myoid cells and fibroblasts of normal mouse testis. The reaction product was observed on the membranes of the endoplasmic reticulum, on the Golgi apparatus and nuclear envelope. The peritubular myoid cells and fibroblasts showed similar ultracytochemical features; the intensity of the enzymatic reaction was suggestive of an important role of the NOS/cGMP enzymatic system in these cells. Some hypotheses on the role of NO in the peritubular myoid cells and fibroblasts are proposed.

NADPH diaphorase activity and nitric oxide synthase immunoreactivity in lordosis-relevant neurons of the ventromedial hypothalamus.

The distribution of the enzymes NADPH diaphorase and nitric oxide synthase in the ventromedial nucleus of the hypothalamus of cycling and ovariectomized/estrogen-treated and control female rats was demonstrated using histochemical and immunocytochemical methods. Serial section analysis of vibratome sections through the entire ventromedial nucleus showed that NADPH diaphorase cellular staining was localized primarily in the ventrolateral subdivision. NADPH diaphorase staining was visible in both neuronal perikarya and processes. Light microscopic immunocytochemistry using affinity-purified polyclonal antibodies to brain nitric oxide synthase revealed a similar pattern of labelling within the ventromedial nucleus and within neurons of the ventrolateral subdivision of the ventromedial nucleus. Control experiments involved omitting the primary antibodies; no labelling was visible under these conditions. Some, but not all, neurons in the ventrolateral subdivision of the ventromedial nucleus contained both NADPH diaphorase and brain nitric oxide synthase as demonstrated by co-localization of these two enzymes in individual cells of this area. That NADPH diaphorase and brain nitric oxide synthase were found in estrogen-binding cells was shown by co-localization of NADPH diaphorase and estrogen receptor and brain nitric oxide synthase and estrogen receptor at the light and ultrastructural levels, respectively. Our studies suggest that brain nitric oxide synthase is present and may be subject to estrogenic influences in lordosis-relevant neurons in the ventrolateral subdivision of the ventromedial nucleus. The hypothalamus is a primary subcortical regulatory center controlling sympathetic function. Therefore, not only is nitric oxide likely to be important for reproductive behavior, but also for the regulation of responses to emotional stress and other autonomic functions.

Expression of nitric oxide synthase in macula densa in streptozotocin diabetic rats.

Renal haemodynamic changes are suggested to be an early sign of diabetic glomerulopathy. The juxtaglomerular apparatus relevant to the renin angiotensin system, known to be the site of nitric oxide (NO) production, is considered to play a role in the regulation of glomerular blood flow. This study was therefore designed to clarify whether in situ expression of nitric oxide synthase (NOS) is altered in the kidney of diabetic rats. Streptozotocin-induced diabetic rats with 6, 8, 12 and 32 weeks diabetes duration and age-matched normal control rats were used. The expression of a constitutive form of NOS (cNOS, neural type) and NADPH diaphorase activity in the renal cortex were studied immunohistochemically and histochemically. Diabetic rats had lower body weight and heavier kidney mass compared to control rats at each time point examined. Mean glomerular surface area was greater in 6, 8 and 12-week diabetic rats compared to age-matched control rats. cNOS reaction was localized in the macula densa and appeared less intense in diabetic rats compared to age-matched control rats. The mean number of macula densa cells positive for cNOS in each glomerulus and in each glomerular area was significantly lower in diabetic rats compared to control rats at any time examined. In contrast, NADPH diaphorase activity was detected in both juxtaglomerular arterioles and macula densa cells. The staining reaction of NADPH diaphorase in the arterioles remained positive but appeared less intense in macula densa cells in diabetic rats. These results suggest that NO production in macula densa cells may be reduced in diabetic rats, modulating the vasodilatory function of afferent arterioles. Further investigation on the changes in inducible NOS as well as endothelial cNOS are necessary to clarify mechanisms of haemodynamic changes in the diabetic kidney.