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Pseudomonas aeruginosa PA1006 is a persulfide-modified protein that is critical for molybdenum homeostasis.

ABSTRACT: A companion manuscript revealed that deletion of the Pseudomonas aeruginosa (Pae) PA1006 gene caused pleiotropic defects in metabolism including a loss of all nitrate reductase activities, biofilm maturation, and virulence. Herein, several complementary approaches indicate that PA1006 protein serves as a persulfide-modified protein that is critical for molybdenum homeostasis in Pae. Mutation of a highly conserved Cys22 to Ala or Ser resulted in a loss of PA1006 activity. Yeast-two-hybrid and a green-fluorescent protein fragment complementation assay (GFP-PFCA) in Pae itself revealed that PA1006 interacts with Pae PA3667/CsdA and PA3814/IscS Cys desulfurase enzymes. Fourier transform ion cyclotron resonance mass spectrometry (FT-ICR-MS) "top-down" analysis of PA1006 purified from Pae revealed that conserved Cys22 is post-translationally modified in vivo in the form a persulfide. Inductively-coupled-plasma (ICP)-MS analysis of ?PA1006 mutant extracts revealed that the mutant cells contain significantly reduced levels of molybdenum compared to wild-type. GFP-PFCA also revealed that PA1006 interacts with several molybdenum cofactor (MoCo) biosynthesis proteins as well as nitrate reductase maturation factor NarJ and component NarH. These data indicate that a loss of PA1006 protein's persulfide sulfur and a reduced availability of molybdenum contribute to the phenotype of a ?PA1006 mutant.

SUBMITTER: Tombline G

PROVIDER: S-EPMC3568144 | biostudies-literature | 2013

REPOSITORIES: biostudies-literature

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Pseudomonas aeruginosa PA1006 is a persulfide-modified protein that is critical for molybdenum homeostasis.

Tombline Gregory G Schwingel Johanna M JM Lapek John D JD Friedman Alan E AE Darrah Thomas T Maguire Michael M Van Alst Nadine E NE Filiatrault Melanie J MJ Iglewski Barbara H BH

PloS one 20130208 2

A companion manuscript revealed that deletion of the Pseudomonas aeruginosa (Pae) PA1006 gene caused pleiotropic defects in metabolism including a loss of all nitrate reductase activities, biofilm maturation, and virulence. Herein, several complementary approaches indicate that PA1006 protein serves as a persulfide-modified protein that is critical for molybdenum homeostasis in Pae. Mutation of a highly conserved Cys22 to Ala or Ser resulted in a loss of PA1006 activity. Yeast-two-hybrid and a g ...[more]

PMID: 23409003

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Project description:The [Re(I)(CO)(3)(4,7-dimethyl-1,10-phenanthroline)(histidine-124)(tryptophan-122)] complex, denoted [Re(I)(dmp)(W122)], of Pseudomonas aeruginosa azurin behaves as a single photoactive unit that triggers very fast electron transfer (ET) from a distant (2 nm) Cu(I) center in the protein. Analysis of time-resolved (ps-?s) IR spectroscopic and kinetics data collected on [Re(I)(dmp)(W122)AzM] (in which M=Zn(II), Cu(II), Cu(I); Az=azurin) and position-122 tyrosine (Y), phenylalanine (F), and lysine (K) mutants, together with excited-state DFT/time-dependent (TD)DFT calculations and X-ray structural characterization, reveal the character, energetics, and dynamics of the relevant electronic states of the [Re(I)(dmp)(W122)] unit and a cascade of photoinduced ET and relaxation steps in the corresponding Re-azurins. Optical population of [Re(I)(imidazole-H124)(CO)(3)]?dmp (1)CT states (CT=charge transfer) is followed by around 110 fs intersystem crossing and about 600 ps structural relaxation to a (3)CT state. The IR spectrum indicates a mixed Re(I)(CO)(3),A?dmp/???(*)(dmp) character for aromatic amino acids A122 (A=W, Y, F) and Re(I)(CO)(3)?dmp metal-ligand charge transfer (MLCT) for [Re(I)(dmp)(K122)AzCu(II)]. In a few ns, the (3)CT state of [Re(I)(dmp)(W122)AzM] establishes an equilibrium with the [Re(I)(dmp(.-))(W122(.+))AzM] charge-separated state, (3)CS, whereas the (3)CT state of the other Y, F, and K122 proteins decays to the ground state. In addition to this main pathway, (3)CS is populated by fs- and ps-W(indole)?Re(II) ET from (1)CT and the initially "hot" (3)CT states, respectively. The (3)CS state undergoes a tens-of-ns dmp(.-)?W122(.+) ET recombination leading to the ground state or, in the case of the Cu(I) azurin, a competitively fast (?30 ns over 1.12?nm) Cu(I)?W(.+) ET, to give [Re(I)(dmp(.-))(W122)AzCu(II)]. The overall photoinduced Cu(I)?Re(dmp) ET through [Re(I)(dmp)(W122)AzCu(I)] occurs over a 2 nm distance in <50 ns after excitation, with the intervening fast (3)CT-(3)CS equilibrium being the principal accelerating factor. No reaction was observed for the three Y, F, and K122 analogues. Although the presence of [Re(dmp)(W122)AzCu(II)] oligomers in solution was documented by mass spectrometry and phosphorescence anisotropy, the kinetics data do not indicate any significant interference from the intermolecular ET steps. The ground-state dmp-indole ?-? interaction together with well-matched W/W(.+) and excited-state [Re(II)(CO)(3)(dmp(.-))]/[Re(I)(CO)(3)(dmp(.-))] potentials that result in very rapid electron interchange and (3)CT-(3)CS energetic proximity, are the main factors responsible for the unique ET behavior of [Re(I)(dmp)(W122)]-containing azurins.

Dataset Information

Pseudomonas aeruginosa PA1006 is a persulfide-modified protein that is critical for molybdenum homeostasis.

Publications

Pseudomonas aeruginosa PA1006 is a persulfide-modified protein that is critical for molybdenum homeostasis.

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