Rhodobacter capsulatus O1sA is a bifunctional enyzme active in both ornithine lipid and phosphatidic acid biosynthesis

The Rhodobacter capsulatus genome contains three genes (olsA [plsC138], plsC316, and plsC3498) that are annotated as lysophosphatidic acid (1-acyl-sn-glycerol-3-phosphate) acyltransferase (AGPAT). Of these genes, olsA was previously shown to be an O-acyltransferase in the second step of ornithine lipid biosynthesis, which is important for optimal steady-state levels of c-type cytochromes (S. Aygun-Sunar, S. Mandaci, H.-G. Koch, I. V. J. Murray, H. Goldfine, and F. Daldal. Mol. Microbiol. 61:418-435, 2006). The roles of the remaining plsC316 and plsC3498 genes remained unknown. In this work, these genes were cloned, and chromosomal insertion-deletion mutations inactivating them were obtained to define their function. Characterization of these mutants indicated that, unlike the Escherichia coli plsC, neither plsC316 nor plsC3498 was essential in R. capsulatus. In contrast, no plsC316 olsA double mutant could be isolated, indicating that an intact copy of either olsA or plsC316 was required for R. capsulatus growth under the conditions tested. Compared to OlsA null mutants, PlsC316 null mutants contained ornithine lipid and had no c-type cytochrome-related phenotype. However, they exhibited slight growth impairment and highly altered total fatty acid and phospholipid profiles. Heterologous expression in an E. coli plsC(Ts) mutant of either R. capsulatus plsC316 or olsA gene products supported growth at a nonpermissive temperature, exhibited AGPAT activity in vitro, and restored phosphatidic acid biosynthesis. The more vigorous AGPAT activity displayed by PlsC316 suggested that plsC316 encodes the main AGPAT required for glycerophospholipid synthesis in R. capsulatus, while olsA acts as an alternative AGPAT that is specific for ornithine lipid synthesis. This study therefore revealed for the first time that some OlsA enzymes, like the enzyme of R. capsulatus, are bifunctional and involved in both membrane ornithine lipid and glycerophospholipid biosynthesis

Substitutions at position 146 of cytochrome b affect drastically the properties of heme b(L) and the Q(o) site of Rhodobacter capsulatus cytochrome bc\u3csub\u3e1\u3c/sub\u3e complex

The cytochrome (cyt) b subunit of ubihydroquinone: cytochrome c oxidoreductase(bc1 complex) contains four invariant glycine (G) residues proposed to be essential for proper packing of the high and low potential (b(H) and b(L)) hemes of the bc1 complex. One of these residues, G146 located in the transmembrane helix C of cyt b of Rhodobacter capsulatus, was substituted with A and V using site-directed mutagenesis, and the effects of these substitutions on the properties of the ubiquinone oxidation (Q(o)) site and heme b(L) of the bc1 complex were analyzed. The mutants G146A and V produced properly assembled but catalytically defective bc1 complexes that are unable to support photosynthetic growth. The steady-state ubihydroquinone: cytochrome c reductase activities of the mutant complexes were about one-tenth of that of a parental strain overproducing the wild-type enzyme. Similarly, their light-activated single turnover rates were significantly lower than those of a wild-type complex. The dark potentiometric titrations revealed no significant changes in the redox midpoint potentials (E(m,7)) of the high (b(H)) and low (b(L)) potential hemes of cyt b in both G146A and V mutants. However, EPR spectroscopy of the [2Fe-2S] cluster of the bc1 complex indicated that the Q(o) site of the mutant enzymes were unoccupied. Moreover, the g(z) signal of heme b(L), but not that of heme b(H), was modified both in G146A and V, suggesting that the geometry of its ligands has been distorted. These findings indicate that this region of cyt b must be well packed around heme b(L) since even a slight increase in the size of the amino acid side chain at position 146 (such as G to A) greatly perturbs the spatial conformation of heme b(L), alters substrate accessibility and binding to the Q(o) site, and renders the bc1 complex inactive

Cu homeostasis in bacteria: The ins and outs

Copper (Cu) is an essential trace element for all living organisms and used as cofactor in key enzymes of important biological processes, such as aerobic respiration or superoxide dismutation. However, due to its toxicity, cells have developed elaborate mechanisms for Cu homeostasis, which balance Cu supply for cuproprotein biogenesis with the need to remove excess Cu. This review summarizes our current knowledge on bacterial Cu homeostasis with a focus on Gram-negative bacteria and describes the multiple strategies that bacteria use for uptake, storage and export of Cu. We furthermore describe general mechanistic principles that aid the bacterial response to toxic Cu concentrations and illustrate dedicated Cu relay systems that facilitate Cu delivery for cuproenzyme biogenesis. Progress in understanding how bacteria avoid Cu poisoning while maintaining a certain Cu quota for cell proliferation is of particular importance for microbial pathogens because Cu is utilized by the host immune system for attenuating pathogen survival in host cells

Ultrafast photochemistry of the bc₁ complex

We present a full investigation of ultrafast light-induced events in the membraneous cytochrome bc 1 complex by transient absorption spectroscopy. This energy-transducing complex harbors four redox-active components per monomer: heme c 1 , two 6-coordinate b-hemes and a [2Fe-2S] cluster. Using excitation of these components in different ratios under various excitation conditions, probing in the full visible range and under three well-defined redox conditions, we demonstrate that for all ferrous hemes of the complex photodissociation of axial ligands takes place and that they rebind in 5-7 ps, as in other 6-coordinate heme proteins, including cytoglobin, which is included as a reference in this study. By contrast, the signals are not consistent with photooxidation of the b hemes. This conclusion contrasts with a recent assessment based on a more limited data set. The binding kinetics of internal and external ligands are indicative of a rigid heme environment, consistent with the electron transfer function. We also report, for the first time, photoactivity of the very weakly absorbing iron-sulfur center. This yields the unexpected perspective of studying photochemistry, initiated by excitation of iron-sulfur clusters, in a range of protein complexes

2007 Molecular & Cellular Bioenergetics (June 17-22, 2007)

This Report describes Molecular & Cellular Bioenergetics

A compilation of mutations located in the cytochrome b subunit of the bacterial and mitochondrial bc1 complex

AbstractIn anticipation of the structure of the bc1 complex which is now imminent, we present here a preliminary compilation of all available cytochrome b mutants that have been isolated or constructed to date both in prokaryotic and eukaryotic species. We have briefly summarized their salient properties with respect to the structure and function of cytochrome b and to the Qo and Qi sites of the bc1 complex. In conjunction with the high resolution structure of the bc1 complex, this database is expected to serve as a useful reference point for the available data and help to focus and stimulate future experimental work in this field