Cardiolipin-based respiratory complex activation in bacteria

Abstract

Anionic lipids play a variety of key roles in membrane function, including functional and structural effects on respiratory complexes. However, little is known about the molecular basis of these lipid-protein interactions. In this study, NarGHI, an anaerobic respiratory complex of Escherichia coli, has been used to investigate the relations in between membrane-bound proteins with phospholipids. Activity of the NarGHI complex is enhanced by anionic phospholipids both in vivo and in vitro. The anionic cardiolipin tightly associates with the NarGHI complex and is the most effective phospholipid to restore functionality of a nearly inactive detergent-solubilized enzyme complex. A specific cardiolipin-binding site is identified on the basis of the available X-ray diffraction data and of site-directed mutagenesis experiment. One acyl chain of cardiolipin is in close proximity to the heme b D center and is responsible for structural adjustments of b D and of the adjacent quinol substrate binding site. Finally, cardiolipin binding tunes the interaction with the quinol substrate. Together, our results provide a molecular basis for the activation of a bacterial respiratory complex by cardiolipin.bioenergetics | EPR spectroscopy | metalloprotein | molybdenum

Q-Site Occupancy Defines Heme Heterogeneity in Escherichia coli Nitrate Reductase A (NarGHI)

“…44 Because NarGHI preferentially uses MQ, which binds to it with a higher affinity over ubiquinone, and better stabilizes menasemiquinone over ubisemiquinone, one therefore expects the component corresponding to the occupied Q-site conformation to correlate most closely with the presence of MQ. 14,34,45 In the case where MQ is absent ( Figure 2B), we observe the most heterogeneity and greatest contribution from the g = 3.18 component. When both MQ and UQ are present, the heterogeneity is reduced, and the g = 3.34 component becomes more prominent.…”

“…14 In this context, the heme b D EPR signal heterogeneity has been interpreted such that the g = 3.18 signal is due to cardiolipin-bound enzyme, and the g = 3.34 signal is due to cardiolipin-free enzyme. 14 To test if cardiolipin does indeed contribute to the heterogeneity, nitrate reductase was expressed semiaerobically in an E. coli strain deficient in its biosynthesis as well as for phosphatidylglycerol (E. coli S330). 16 As shown the Figure 2A …”

“…10 Heterogeneity of heme b D has been reported in a mutant unable to synthesize Mo-bisPGD, 13 resulting in the appearance of two components with g z values of approximately 3.35 and 3.21. AriasCartin et al 14 also reported that heme b D can exist in two forms, one with a g z of approximately 3.20 and the other with a g z of approximately 3.35. Extraction of hydrophobic components using dodecylmaltoside (DDM) resulted in diminution of the g = 3.20 component and retention of the g = 3.35 component.…”

Determination of the Proton Environment of High Stability Menasemiquinone Intermediate in Escherichia coli Nitrate Reductase A by Pulsed EPR

“…Thus, we tentatively proposed that a water-mediated interaction is formed between MSQ D (or USQ D ) and Lys-86, consistent with the latter being involved in reactivity toward quinols (13). Moreover, we have recently shown that a cardiolipin molecule specifically bound to the complex is necessary for quinol substrate fixation at the Q D site, probably through the action of one of its acyl chains located in the vicinity of His-66 (15). Clearly, additional information is required to improve our understanding of the semiquinone binding mode in the Q D site and of its functional tuning by the protein environment.…”

“…The use of ESEEM and HYSCORE (hyperfine sublevel correlation) spectroscopies on either the wild-type enzyme or the enzyme uniformly enriched with 15 N nuclei provided direct evidence for nitrogen ligation to MSQ D and USQ D . On the basis of the direct determination of the quadrupolar parameters of the corresponding interacting 14 N by S-band (ϳ3 GHz) HYSCORE experiments, we assigned the latter to an N ␦ imidazole nitrogen and proposed it to arise from the heme b D axial ligand His-66 (13).…”