The Tat Substrate CueO Is Transported in an Incomplete Folding State.

In Escherichia coli, cytoplasmic copper ions are toxic to cells even at the lowest concentrations. As a defense strategy, the cuprous oxidase CueO is secreted into the periplasm to oxidize the more membrane-permeable and toxic Cu(I) before it can enter the cytoplasm. CueO itself is a multicopper oxidase that requires copper for activity. Because it is transported by the twin-arginine translocation (Tat) pathway, which transports folded proteins, a requirement for cofactor assembly before translocation has been discussed. Here we show that CueO is transported as an apo-protein. Periplasmic CueO was readily activated by the addition of copper ions in vitro or under copper stress conditions in vivo Cytoplasmic CueO did not contain copper, even under copper stress conditions. In vitro Tat transport proved that the cofactor assembly was not required for functional Tat transport of CueO. Due to the post-translocational activation of CueO, this enzyme contributes to copper resistance not only by its cuprous oxidase activity but also by chelation of copper ions before they can enter the cytoplasm. Apo-CueO was indistinguishable from holo-CueO in terms of secondary structural elements. Importantly, the binding of copper to apo-CueO greatly stabilized the protein, indicating a transformation from an open or flexible domain arrangement with accessible copper sites to a closed structure with deeply buried copper ions. CueO is thus the first example for a natural Tat substrate of such incomplete folding state. The Tat system may need to transport flexibly folded proteins in any case when cofactor assembly or quaternary structure formation occurs after transport.

Homologous expression of the nrdF gene of Corynebacterium ammoniagenes strain ATCC 6872 generates a manganese-metallocofactor (R2F) and a stable tyrosyl radical (Y˙) involved in ribonucleotide reduction.

Ribonucleotide reduction, the unique step in the pathway to DNA synthesis, is catalyzed by enzymes via radical-dependent redox chemistry involving an array of diverse metallocofactors. The nucleotide reduction gene (nrdF) encoding the metallocofactor containing small subunit (R2F) of the Corynebacterium ammoniagenes ribonucleotide reductase was reintroduced into strain C. ammoniagenes ATCC 6872. Efficient homologous expression from plasmid pOCA2 using the tac-promotor enabled purification of R2F to homogeneity. The chromatographic protocol provided native R2F with a high ratio of manganese to iron (30:1), high activity (69 µmol 2'-deoxyribonucleotide·mg⁻¹ ·min⁻¹) and distinct absorption at 408 nm, characteristic of a tyrosyl radical (Y˙), which is sensitive to the radical scavenger hydroxyurea. A novel enzyme assay revealed the direct involvement of Y˙ in ribonucleotide reduction because 0.2 nmol 2'-deoxyribonucleotide was formed, driven by 0.4 nmol Y˙ located on R2F. X-band electron paramagnetic resonance spectroscopy demonstrated a tyrosyl radical at an effective g-value of 2.004. Temperature dependent X/Q-band EPR studies revealed that this radical is coupled to a metallocofactor. Similarities of the native C. ammoniagenes ribonucleotide reductase to the in vitro activated Escherichia coli class Ib enzyme containing a dimanganese(III)-tyrosyl metallocofactor are discussed.

A tyrosyl-dimanganese coupled spin system is the native metalloradical cofactor of the R2F subunit of the ribonucleotide reductase of Corynebacterium ammoniagenes.

The X-ray crystallographic structure of the native R2F subunit of the ribonucleotide reductase (RNR) of Corynebacterium ammoniagenes ATCC 6872 is reported, with a resolution of 1.36 A. The metal site contains an oxo/hydroxo-bridged manganese dimer, located near a tyrosine residue (Y115). The coordination of the manganese dimer and its distance to a nearby tyrosine residue resemble the di-iron metalloradical cofactor of class I RNR from Escherichia coli . Multifrequency EPR measurements of the highly active C. ammoniagenes R2F subunit show that the metal site contains a ferromagnetically exchange-coupled Mn(III)Mn(III) dimer weakly coupled to a tyrosyl radical. A mechanism for the metalloradical cofactor (Mn(III)Mn(III)Y(*)) generation is proposed. H(2)O(2) (HO(2)(-)) instead of O(2) is hypothesized as physiological oxidant for the Mn dimer which in turn oxidizes the tyrosine Y115. Changes in the ligand sphere of both manganese ions during metalloradical generation direct the complex formation of this cofactor, disfavoring alternate reaction pathways such as H(2)O(2) dismutation, as observed for manganese catalase, a structural analogue of the R2F metal site. The presented results demonstrate the importance of manganese for radical formation in this RNR and confirm the assignment of this enzyme to class Ib.

Crystallization and preliminary X-ray analysis of the small subunit (R2F) of native ribonucleotide reductase from Corynebacterium ammoniagenes.

Ribonucleotide reduction, the unique step in DNA-precursor biosynthesis, involves radical-dependent redox chemistry and diverse metallo-cofactors. The metallo-cofactor (R2F) encoded by the nrdF (nucleotide reduction) gene in Corynebacterium ammoniagenes ATCC 6872 was isolated after homologous expression and a new crystal form of ribonucleotide reductase R2F was obtained. R2F was crystallized at 277 K using the vapour-diffusion method with PEG as the precipitating agent. A data set was collected to 1.36 A resolution from a single crystal at 100 K using synchrotron radiation. The crystal belonged to space group C2, with unit-cell parameters a = 96.21, b = 87.68, c = 83.25 A, beta = 99.29 degrees. The crystal contained two molecules per asymmetric unit, with a Matthews coefficient (V(M)) of 2.69 A(3) Da(-1); the solvent content was estimated to be 54.3%. X-ray fluorescence spectroscopy and MAD diffraction data indicated the presence of manganese in the molecule and the absence of iron.

Electron paramagnetic resonance (EPR) spectroscopy of the stable-free radical in the native metallo-cofactor of the manganese-ribonucleotide reductase (Mn-RNR) of Corynebacterium glutamicum.

Ribonucleotide reductases (RNR; EC 1.17.4.1) provide the 2'-deoxyribonucleotides for DNA replication of proliferating cells by a uniform radical mechanism using diverse metals. The native metallo-cofactor of the Corynebacterium glutamicum RNR contains manganese and is sensitive to EDTA and radical scavengers. Hybrid holoenzymes, capable of ribonucleotide reduction, were composed of the small manganese-containing (R2F) and the large catalytic subunit (R1E) from either of the two corynebacterial RNRs. A synthetic peptide deduced from the C-terminal region of the nrdF gene inhibited the C. glutamicum-RNR non-competitively and cross-reacted with the C. ammoniagenes-RNR. The C. glutamicum-R2F has a saturable organic radical signal at g=2.005 detected by electron paramagnetic resonance (EPR) spectroscopy and shows a distinct absorption at 408 nm indicative of a tyrosyl-like organic radical (Y.). Quantification of the metal content revealed 0.06 mol Fe but 0.8 mol Mn per mol R2F-monomer and would thus assign two manganese atoms bound to the dimeric metallo-cofactor, while a distinct enzymatic activity (32 micromol x mg(-1) x min(-1)) was observed in the biochemical complementation assay. Divergence of the C. glutamicum-RNR studied here from the prototypical Salmonella typhimurium class 1b enzyme and the Chlamydia trachomatis class Ic enzyme is discussed below.