No abstract

The crystal structure of the anaerobic complex of Pseudomonas putida protocatechuate 3,4-dioxygenase (3,4-PCD) bound with the alternative substrate, 3,4-dihydroxyphenylacetate (HPCA), is reported at 2.4 A resolution and refined to an R factor of 0.17. Formation of the active site Fe(III).HPCA chelated complex causes the endogenous axial tyrosinate, Tyr447 (147beta), to dissociate from the iron and rotate into an alternative orientation analogous to that previously observed in the anaerobic 3,4-PCD.3,4-dihydroxybenzoate complex (3, 4-PCD.PCA) [Orville, A. M., Lipscomb, J. D., & Ohlendorf, D. H. (1997) Biochemistry 36, 10052-10066]. Two orientations of the aromatic ring of HPCA related by an approximate 180 degrees rotation within the active site are consistent with the electron density. Resonance Raman (rR) spectroscopic data from Brevibacteriumfuscum 3,4-PCD.HPCA complex in solution reveals low frequency rR vibrational bands between 500 and 650 cm-1 as well as a band at approximately 1320 cm-1 which are diagnostic of a HPCA. Fe(III) chelate complex. 18O labeling of HPCA at either the C4 or C3 hydroxyl group unambiguously establishes the vibrational coupling modes associated with the five-membered chelate ring system. Analysis of these data suggests that the Fe(III)-HPCAO4 bond is shorter than the Fe(III)-HPCAO3 bond. This consequently favors the model for the crystal structure in which the C3 phenolic function occupies the Fe3+ ligand site opposite the endogenous ligand Tyr408(Oeta) (108beta). This is essentially the same binding orientation as proposed for PCA in the crystal structure of the anaerobic 3,4-PCD.PCA complex based solely on direct modeling of the 2Fo - Fc electron density and suggests that this is the conformation required for catalysis.

The small subunit of Escherichia coli ribonucleotide reductase (R2) is a homodimeric (betabeta) protein, in which each beta-peptide contains a diiron cluster composed of two inequivalent iron sites. R2 is capable of reductively activating O(2) to produce a stable tyrosine radical (Y122*), which is essential for production of deoxyribonucleotides on the larger R1 subunit. In this work, the paramagnetic Mn(II) ion is used as a spectroscopic probe to characterize the assembly of the R2 site with EPR spectroscopy. Upon titration of Mn(II) into samples of apoR2, we have been able to quantitatively follow three species (aquaMn(II), mononuclear Mn(II)R2, and dinuclear Mn(2)(II)R2) and fit each to a sequential two binding site model. As previously observed for Fe(II) binding within apoR2, one of the sites has a greater binding affinity relative to the other, K(1) = (5.5 +/- 1.1) x 10(5) M(-)(1) and K(2) = (3.9 +/- 0.6) x 10(4) M(-)(1), which are assigned to the B and A sites, respectively. In multiple titrations, only one dinuclear Mn(2)(II)R2 site was created per homodimer of R2, indicating that only one of the two beta-peptides of R2 is capable of binding Mn(II) following addition of Mn(II) to apoR2. Under anaerobic conditions, addition of only 2 equiv of Fe(II) to R2 (Fe(2)(II)R2) completely prevented the formation of any bound MnR2 species. Upon reaction of this sample with O(2) in the presence of Mn(II), both Y122* and Mn(2)(II)R2 were produced in equal amounts. Previous stopped-flow absorption spectroscopy studies have indicated that apoR2 undergoes a protein conformational change upon binding of metal (Tong et al. J. Am. Chem. Soc. 1996, 118, 2107-2108). On the basis of these observations, we propose a model for R2 metal incorporation that invokes an allosteric interaction between the two beta-peptides of R2. Upon binding the first equiv of metal to a beta-peptide (beta(I)), the aforementioned protein conformational change prevents metal binding in the adjacent beta-peptide (beta(II)) approximately 25 A away. Furthermore, we show that metal incorporation into beta(II) occurs only during the O(2) activation chemistry of the beta(I)-peptide. This is the first direct evidence of an allosteric interaction between the two beta-peptides of R2. Furthermore, this model can explain the generally observed low Fe occupancy of R2. We also demonstrate that metal uptake and this newly observed allosteric effect are buffer dependent. Higher levels of glycerol cause loss of the allosteric effect. Reductive cycling of samples in the presence of Mn(II) produced a novel mixed metal Fe(III)Mn(III)R2 species within the active site of R2. The magnitude of the exchange coupling (J) determined for both the Mn(2)(II)R2 and Fe(III)Mn(III)R2 species was determined to be -1.8 +/- 0.3 and -18 +/- 3 cm(-)(1), respectively. Quantitative spectral simulations for the Fe(III)Mn(III)R2 and mononuclear Mn(II)R2 species are provided. This work represents the first instance where both X- and Q-band simulations of perpendicular and parallel mode spectr...