Marly K. Eidsness

Crystal structure of rubredoxin from Pyrococcus furiosus at 0.95 Å resolution, and the structures of N-terminal methionine and formylmethionine variants of Pf Rd. Contributions of N-terminal interactions to thermostability

Abstract The high-resolution crystal structure of the small iron-sulfur protein rubredoxin (Rd) from the hyperthermophilic archeon Pyrococcus furiosus (Pf) is reported in this paper, together with those of its methionine ([_0M]Pf Rd) and formylmethionine (f[_0M]Pf Rd) variants. These studies were conducted to assess the consequences of the presence or absence of a salt bridge between the amino terminal nitrogen of Ala1 and the side chain of Glu14 to the structure and stability of this rubredoxin. The structure of wild-type Pf Rd was solved to a resolution of 0.95 Å and refined by full-matrix least-squares techniques to a crystallographic agreement factor of 12.8% [F>2σ(F) data, 25 617 reflections], while those of the [_0M]Pf and f[_0M]Pf Rd variants were solved at slightly lower resolutions (1.1 Å, R=11.5%, 17 213 reflections; 1.2 Å, R=13.7%, 12 478 reflections, respectively). The quality of the data was such that about half of the hydrogen atoms of the protein were clearly visible. All three structures were ultimately refined using the program SHELXL-93 with anisotropic atomic displacement parameters for all non-hydrogen protein atoms, and calculated hydrogen positions included but not refined. In this paper we also report thermostability data for all three forms of Pf Rd, and show that they follow the sequence wild-type >[_0M]Pf>formyl[_0M]Pf. Comparison of the three Pf Rd structures in the N-terminal region show that the structures of wild-type Pf Rd and f[_0M]Pf are rather similar, while that of [_0M]Pf Rd shows a number of additional hydrogen bonds involving the extra methionine group. While the salt bridge between the Ala1 amino group and the Glu14 carboxylate is not the primary determinant of the thermostability of Pf Rd, alterations to the amino terminus do have a moderate influence on the thermostability of this protein.

Leucine 41 is a gate for water entry in the reduction of Clostridium pasteurianum rubredoxin

Biological electron transfer is an efficient process even though the distances between the redox moieties are often quite large. It is therefore of great interest to gain an understanding of the physical basis of the rates and driving forces of these reactions. The structural relaxation of the protein that occurs upon change in redox state gives rise to the reorganizational energy, which is important in the rates and the driving forces of the proteins involved. To determine the structural relaxation in a redox protein, we have developed methods to hold a redox protein in its final oxidation state during crystallization while maintaining the same pH and salt conditions of the crystallization of the protein in its initial oxidation state. Based on 1.5 Å resolution crystal structures and molecular dynamics simulations of oxidized and reduced rubredoxins (Rd) from Clostridium pasteurianum (Cp), the structural rearrangements upon reduction suggest specific mechanisms by which electron transfer reactions of rubredoxin should be facilitated. First, expansion of the [Fe—S] cluster and concomitant contraction of the NH • • • S hydrogen bonds lead to greater electrostatic stabilization of the extra negative charge. Second, a gating mechanism caused by the conformational change of Leucine 41, a nonpolar side chain, allows transient penetration of water molecules, which greatly increases the polarity of the redox site environment and also provides a source of protons. Our method of producing crystals of Cp Rd from a reducing solution leads to a distribution of water molecules not observed in the crystal structure of the reduced Rd from Pyrococcus furiosus. How general this correlation is among redox proteins must be determined in future work. The combination of our high‐resolution crystal structures and molecular dynamics simulations provides a molecular picture of the structural rearrangement that occurs upon reduction in Cp rubredoxin.

Prediction of Reduction Potential Changes in Rubredoxin: A Molecular Mechanics Approach

Predicting the effects of mutation on the reduction potential of proteins is crucial in understanding how reduction potentials are modulated by the protein environment. Previously, we proposed that an alanine vs. a valine at residue 44 leads to a 50-mV difference in reduction potential found in homologous rubredoxins because of a shift in the polar backbone relative to the iron site due to the different side-chain sizes. Here, the aim is to determine the effects of mutations to glycine, isoleucine, and leucine at residue 44 on the structure and reduction potential of rubredoxin, and if the effects are proportional to side-chain size. Crystal structure analysis, molecular mechanics simulations, and experimental reduction potentials of wild-type and mutant Clostridium pasteurianum rubredoxin, along with sequence analysis of homologous rubredoxins, indicate that the backbone position relative to the redox site as well as solvent penetration near the redox site are both structural determinants of the reduction potential, although not proportionally to side-chain size. Thus, protein interactions are too complex to be predicted by simple relationships, indicating the utility of molecular mechanics methods in understanding them.

Recombinant Escherichia coli biotin synthase is a [2Fe–2S]2+ protein in whole cells

EPR and Mössbauer spectroscopies have been used to determine the type and properties of the iron–sulfur clusters present in homologously expressed recombinant Escherichia coli BioB in whole cells prior to purification. Difference EPR spectra of samples of whole cells from a strain over‐expressing E. coli BioB and a strain containing the same plasmid but without the bioB insertion showed an axial S=1/2 resonance that was attributed to the [2Fe–2S]+ cluster of the E. coli iron–sulfur cluster assembly 2Fe ferredoxin, based on principal g‐values, linewidths and relaxation behavior. Comparison of the Mössbauer spectra of whole cells with and without the bioB insertion revealed that the E. coli cells with over‐expressed BioB contain an additional species that exhibits a spectrum identical to that of the [2Fe–2S]2+ cluster in purified recombinant BioB. The concentration of this [2Fe–2S]2+ species in the whole cell sample was quantified using a Mössbauer standard and found to be approximately 260 μM, which was comparable to the BioB protein concentration estimated for the cell paste. The results demonstrate that the [2Fe–2S]2+ cluster found in purified samples of recombinant BioB is not an artifact of the protein purification procedure, and indicate that recombinant BioB is over‐expressed in an inactive form during aerobic growth.

The Use of X-Ray Absorption Spectroscopy for Detection of Metal-Metal Interactions. Application to Copper-Containing Enzymes

Abstract The use of the extended x-ray absorption fine structure (EXAFS) technique for the identification of metal-metal distances in biomacromolecules is discussed. The Cu EXAFS data for a number of structurally characterized copper-containing bi-nuclear complexes are analyzed to determine the viability of detecting a Cu[sbnd]M (M=Cu, Fe) scattering interaction at ∼3 A in the presence of Cu[sbnd]C interactions at approximately the same distance (deriving from the outer-shell atoms of heterocyclic ligands). The techniques developed are then applied to the oxidized and reduced forms of the copper-containing enzyme dopamine β-hydroxylase. Although in principle the EXAFS technique has the ability to distinguish C from M scatterers, based on differences in the backscattering amplitude and phase, in the absence of a priori knowledge of the distribution of C atoms about the Cu site, often no unambiguous determination of the presence or absence of a Cu[sbnd]M interaction can be made. It is suggested that caution...

Crystallographic studies of V44 mutants of Clostridium pasteurianum rubredoxin: Effects of side‐chain size on reduction potential

Understanding the structural origins of differences in reduction potentials is crucial to understanding how various electron transfer proteins modulate their reduction potentials and how they evolve for diverse functional roles. Here, the high‐resolution structures of several Clostridium pasteurianum rubredoxin (Cp Rd) variants with changes in the vicinity of the redox site are reported in order to increase this understanding. Our crystal structures of [V44L] (at 1.8 Å resolution), [V44A] (1.6 Å), [V44G] (2.0 Å) and [V44A, G45P] (1.5 Å) Rd (all in their oxidized states) show that there is a gradual decrease in the distance between Fe and the amide nitrogen of residue 44 upon reduction in the size of the side chain of residue 44; the decrease occurs from leucine to valine, alanine or glycine and is accompanied by a gradual increase in their reduction potentials. Mutation of Cp Rd at position 44 also changes the hydrogen‐bond distance between the amide nitrogen of residue 44 and the sulfur of cysteine 42 in a size‐dependent manner. Our results suggest that residue 44 is an important determinant of Rd reduction potential in a manner dictated by side‐chain size. Along with the electric dipole moment of the 43‐44 peptide bond and the 44–42 NHS type hydrogen bond, a modulation mechanism for solvent accessibility through residue 41 might regulate the redox reaction of the Rds. Proteins 2004.

Contribution of the [FeII(SCys)4] site to the thermostability of rubredoxins

The thermostabilities of Fe2+ ligation in rubredoxins (Rds) from the hyperthermophile Pyrococcus furiosus (Pf) and the mesophiles Clostridium pasteurianum (Cp) and Desulfovibrio vulgaris (Dv) were compared. Residue 44 forms an NH...S(Cys) hydrogen bond to one of the cysteine ligands to the [Fe(SCys)4] site, and substitutions at this location affect the redox properties of the [Fe(SCys)4] site. Both Pf Rd and Dv Rd have an alanine residue at position 44, whereas Cp Fd has a valine residue. Wild-type proteins were examined along with V44A and A44V “exchange” mutants of Cp and Pf Rds, respectively, in order to assess the effects of the residue at position 44 on the stability of the [Fe(SCys)4] site. Stability of iron ligation was measured by temperature-ramp and fixed-temperature time course experiments, monitoring iron release in both the absence and presence of more thiophilic metals (Zn2+, Cd2+) and over a range of pH values. The thermostability of the polypeptide fold was concomitantly measured by fluorescence, circular dichroism, and 1H NMR spectroscopies. The A44V mutation strongly lowered the stability of the [FeII(SCys)4] site in Pf Rd, whereas the converse V44A mutation of Cp Rd significantly raised the stability of the [FeII(SCys)4] site, but not to the levels measured for wild-type Dv Rd. The region around residue 44 is thus a significant contributor to stability of iron coordination in reduced Rds. This region, however, made only a minor contribution to the thermostability of the protein folding, which was found to be higher for hyperthermophilic versus mesophilic Rds, and largely independent of the residue at position 44. These results, together with our previous studies, show that localized charge density, solvent accessibility, and iron site/backbone interactions control the thermostability of the [Fe(SCys)4] site. The iron site thermostability does make a minor contribution to the overall Rd thermostability. From a mechanistic standpoint, we also found that attack of displacing ions (H+, Cd2+) on the Cys42 sulfur ligand at the [Fe(SCys)4] site occurs through the V8 side and not the V44 side of the iron site.