A pathway for cytochrome c maturation (Ccm) in bacteria, archaea and eukaryotes (mitochondria) requires the genes encoding eight membrane proteins (CcmABCDEFGH). The CcmABCDE proteins are proposed to traffic haem to the cytochrome c synthetase (CcmF/H) for covalent attachment to cytochrome c by unknown mechanisms. For the first time, we purify pathway complexes with trapped haem to elucidate the molecular mechanisms of haem binding, trafficking and redox control. We discovered an early step in trafficking that involves oxidation of haem (to Fe3+), yet the final attachment requires reduced haem (Fe2+). Surprisingly, CcmF is a cytochrome b with a haem never before realized, and in vitro, CcmF functions as a quinol:haem oxidoreductase. Thus, this ancient pathway has conserved and orchestrated mechanisms for trafficking, storing and reducing haem, which assure its use for cytochrome c synthesis even in limiting haem (iron) environments and reducing haem in oxidizing environments.
Glutathione biosynthesis is a key component in the network of plant stress responses that counteract oxidative damage and maintain intracellular redox environment. Using a combination of mass spectrometry and site-directed mutagenesis, we examined the response of Arabidopsis thaliana glutamate-cysteine ligase (GCL) to changes in redox environment. Mass spectrometry identified two disulfide bonds (Cys186-Cys406 and Cys349-Cys364) in GCL. Mutation of either Cys-349 or Cys-364 to a Ser reduced reaction rate by twofold, but substitution of a Ser for either Cys-186 or Cys-406 decreased activity by 20-fold and abrogated the response to changes in redox environment. Redox titrations show that the regulatory disulfide bond has a midpoint potential comparable with other known redox-responsive plant proteins. Mutation of Cys-102, Cys-251, Cys-349, or Cys-364 did not alter the response to redox environment, indicating that modulation of activity depends on the Cys186-Cys406 disulfide bond. In vivo analysis of GCL in Arabidopsis root extracts revealed that multiple oxidative stresses altered the distribution of oxidized (active) and reduced (inactive) enzyme and that this change correlated with increased GCL activity. The thiol-based regulation of GCL provides a posttranslational mechanism for modulating enzyme activity in response to in vivo redox environment and suggests a role for oxidative signaling in the maintenance of glutathione homeostasis in plants.
The pyrimidine nucleotide biosynthesis (pyr) operon in Bacillus subtilis is regulated by transcriptional attenuation. The PyrR protein binds in a uridine nucleotide-dependent manner to three attenuation sites at the 5'-end of pyr mRNA. PyrR binds an RNA-binding loop, allowing a terminator hairpin to form and repressing the downstream genes. The binding of PyrR to defined RNA molecules was characterized by a gel mobility shift assay. Titration indicated that PyrR binds RNA in an equimolar ratio. PyrR bound more tightly to the binding loops from the second (BL2 RNA) and third (BL3 RNA) attenuation sites than to the binding loop from the first (BL1 RNA) attenuation site. PyrR bound BL2 RNA 4-5-fold tighter in the presence of saturating UMP or UDP and 150- fold tighter with saturating UTP, suggesting that UTP is the more important co-regulator. The minimal RNA that bound tightly to PyrR was 28 nt long. Thirty-one structural variants of BL2 RNA were tested for PyrR binding affinity. Two highly conserved regions of the RNA, the terminal loop and top of the upper stem and a purine-rich internal bulge and the base pairs below it, were crucial for tight binding. Conserved elements of RNA secondary structure were also required for tight binding. PyrR protected conserved areas of the binding loop in hydroxyl radical footprinting experiments. PyrR likely recognizes conserved RNA sequences, but only if they are properly positioned in the correct secondary structure.
Bacillus subtilis PyrR has been shown to mediate transcriptional attenuation at three separate sites within the pyrimidine nucleotide biosynthetic (pyr) operon. Molecular genetic evidence suggests that regulation is achieved by PyrR binding to pyr mRNA. PyrR is also a uracil phosphoribosyltransferase (UPRTase). Recombinant PyrR was expressed in Escherichia coli, purified to homogeneity, physically and chemically characterized, and examined with respect to both of these activities. Mass spectroscopic characterization of PyrR demonstrated a monomeric mass of 20,263 Da. Gel filtration chromatography showed the native mass of PyrR to be dependent on protein concentration and suggested a rapid equilibrium between dimeric and hexameric forms. The UPRTase activity of PyrR has a pH optimum of 8.2. The K m value for uracil is very pH-dependent; the K m for uracil at pH 7.7 is 990 ؎ 114 M, which is much higher than for most UPRTases and may account for the low physiological activity of PyrR as a UPRTase. Using an electrophoretic mobility shift assay, PyrR was shown to bind pyr RNA that includes sequences from its predicted binding site in the second attenuator region. Binding of PyrR to pyr RNA was specific and UMP-dependent with apparent K d values of 10 and 220 nM in the presence and absence of UMP, respectively. The concentration of UMP required for half-maximal stimulation of binding of PyrR to RNA was 6 M. The results support a model for the regulation of pyr transcription whereby termination is governed by the UMP-dependent binding of PyrR to pyr RNA and provide purified and characterized PyrR for detailed biochemical studies of RNA binding and transcriptional attenuation.The Bacillus subtilis pyrimidine biosynthetic (pyr) operon encodes all of the enzymes for the de novo biosynthesis of UMP and two additional cistrons encoding a uracil permease and the regulatory protein PyrR (1-4). On the basis of molecular genetic evidence it was proposed that PyrR regulates pyr expression through a transcriptional attenuation mechanism that acts at three separate sites within the operon, which are located in the 5Ј-untranslated leader, between the first (pyrR) and second (pyrP) genes, and between the second and third (pyrB) genes of the operon (3, 5). PyrR is proposed to regulate the ratio of terminated to readthrough transcripts at each attenuation site by permitting the formation of a -independent transcription terminator when exogenous pyrimidines are available. The binding of PyrR to pyr mRNA interferes with the formation of an alternative upstream stem-loop structure, the antiterminator, which is otherwise kinetically and thermodynamically favored. The presence of a conserved sequence within the 5Ј-stem of each antiterminator suggested a site within the pyr mRNA for interaction with PyrR (3); this site is the locus of several cis-acting mutants in the first pyr attenuator which are deficient in repression by pyrimidines (6).In addition, PyrR functions as a novel uracil phosphoribosyltransferase (UPRTase), 1 catalyzing the f...
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