Quantum chemical comparison of vertical, adiabatic, and 0-0 excitation energies: the PYP and GFP chromophores.

A number of benchmark studies investigating the performance of quantum chemical methods for calculating vertical excitation energies are today available in the literature. However, less established is the variation between methods in their estimates of the differences between vertical, adiabatic, and 0-0 excitation energies. To this end, such excitation energies are here calculated for the bright S(1) states of the anionic chromophores of the photoactive yellow protein (PYP) and the green fluorescent protein (GFP) in the gas phase using configuration interaction singles, complete active space self-consistent field, coupled-cluster singles and doubles, and time-dependent density functional theory methods. Although the estimates of the excitation energies vary by more than 1 eV between the methods, the differences between the different types of excitation energies are found to be relatively method-insensitive, varying by ~0.1 eV only for these particular chromophores. Specifically, the adiabatic energies are uniformly 0.10-0.17 (PYP) and 0.06-0.17 eV (GFP) lower than the vertical energies, and the 0-0 energies are similarly 0.09-0.14 (PYP) and 0.07-0.17 eV (GFP) lower than the adiabatic energies.

Orientation of the tyrosyl radical in Salmonella typhimurium class Ib ribonucleotide reductase determined by high field EPR of R2F single crystals.

The R2 protein of class I ribonucleotide reductase (RNR) generates and stores a tyrosyl radical, located next to a diferric iron center, which is essential for ribonucleotide reduction and thus DNA synthesis. X-ray structures of class Ia and Ib proteins from various organisms served as bases for detailed mechanistic suggestions. The active site tyrosine in R2F of class Ib RNR of Salmonella typhimurium is located at larger distance to the diiron site, and shows a different side chain orientation, as compared with the tyrosine in R2 of class Ia RNR from Escherichia coli. No structural information has been available for the active tyrosyl radical in R2F. Here we report on high field EPR experiments of single crystals of R2F from S. typhimurium, containing the radical Tyr-105*. Full rotational pattern of the spectra were recorded, and the orientation of the g-tensor axes were determined, which directly reflect the orientation of the radical Tyr-105* in the crystal frame. Comparison with the orientation of the reduced tyrosine Tyr-105-OH from the x-ray structure reveals a rotation of the tyrosyl side chain, which reduces the distance between the tyrosyl radical and the nearest iron ligands toward similar values as observed earlier for Tyr-122* in E. coli R2. Presence of the substrate binding subunit R1E did not change the EPR spectra of Tyr-105*, indicating that binding of R2E alone induces no structural change of the diiron site. The present study demonstrates that structural and functional information about active radical states can be obtained by combining x-ray and high-field-EPR crystallography.

The first holocomplex structure of ribonucleotide reductase gives new insight into its mechanism of action.

Ribonucleotide reductase is an indispensable enzyme for all cells, since it catalyses the biosynthesis of the precursors necessary for both building and repairing DNA. The ribonucleotide reductase class I enzymes, present in all mammals as well as in many prokaryotes and DNA viruses, are composed mostly of two homodimeric proteins, R1 and R2. The reaction involves long-range radical transfer between the two proteins. Here, we present the first crystal structure of a ribonucleotide reductase R1/R2 holocomplex. The biological relevance of this complex is based on the binding of the R2 C terminus in the hydrophobic cleft of R1, an interaction proven to be crucial for enzyme activity, and by the fact that all conserved amino acid residues in R2 are facing the R1 active sites. We suggest that the asymmetric R1/R2 complex observed in the 4A crystal structure of Salmonella typhimurium ribonucleotide reductase represents an intermediate stage in the reaction cycle, and at the moment of reaction the homodimers transiently form a tight symmetric complex.

Crystal structure of the biologically active form of class Ib ribonucleotide reductase small subunit from Mycobacterium tuberculosis.

Two nrdF genes of Mycobacterium tuberculosis code for different R2 subunits of the class Ib ribonucleotide reductase (RNR). The proteins are denoted R2F-1 and R2F-2 having 71% sequence identity. The R2F-2 subunit forms the biologically active RNR complex with the catalytic R1E-subunit. We present the structure of the reduced R2F-2 subunit to 2.2 A resolution. Comparison of the R2F-2 structure with a model of R2F-1 suggests that the important differences are located at the C-terminus. We found that within class Ib, the E-helix close to the iron diiron centre has two preferred conformations, which cannot be explained by the redox-state of the diiron centre. In the R2F-2 structure, we also could see a mobility of alphaE in between the two conformations.

Structure of the large subunit of class Ib ribonucleotide reductase from Salmonella typhimurium and its complexes with allosteric effectors.

The three-dimensional structure of the large subunit of the first member of a class Ib ribonucleotide reductase, R1E of Salmonella typhimurium, has been determined in its native form and together with three allosteric effectors. The enzyme contains the characteristic ten-stranded alpha/beta-barrel with catalytic residues at a finger loop in its center and with redox-active cysteine residues at two adjacent barrel strands. Structures where the redox-active cysteine residues are in reduced thiol form and in oxidized disulfide form have been determined revealing local structural changes. The R1E enzyme differs from the class Ia enzyme, Escherichia coli R1, by not having an overall allosteric regulation. This is explained from the structure by differences in the N-terminal domain, which is about 50 residues shorter and lacks the overall allosteric binding site. R1E has an allosteric substrate specificity regulation site and the binding site for the nucleotide effectors is located at the dimer interface similarly as for the class Ia enzymes. We have determined the structures of R1E in the absence of effectors and with dTTP, dATP and dCTP bound. The low affinity for ATP at the specificity site is explained by a tyrosine, which hinders nucleotides containing a 2'-OH group to bind.

Expression and preliminary crystallographic studies of R1E, the large subunit of ribonucleotide reductase from Salmonella typhimurium.

The nrdE gene product R1E, the large subunit of the class 1b Salmonella typhimurium ribonucleotide reductase, has been overexpressed, purified and crystallized. Initially, the protein crystallized in two orthorhombic space groups, C222(1) and P2(1)2(1)2, using tartrate and PEG 6000 as precipitants, respectively. Better diffracting crystals belonging to the tetrahedral space group P4(3)2(1)2 were obtained using sodium malonate as precipitant. The P4(3)2(1)2 crystals could only be obtained after seeding from a drop containing C222(1) crystals grown in sodium tartrate. Thus, streak-seeding resulted in crystals of a supergroup to C222(1). Data to 2.8 A resolution have been collected on the P4(3)2(1)2 crystals which contained one R1E subunit in the asymmetric unit.