Modulation of the Redox Potential of the [Fe(SCys)₄] Site in Rubredoxin by the Orientation of a Peptide Dipole

Abstract

Rubredoxins (Rds) may be separated into two classes based upon the correlation of their reduction potentials with the identity of residue 44; those with Ala44 have reduction potentials that are approximately 50 mV higher than those with Val44. The smaller side chain volume occupied by Ala44 relative to that occupied by Val44 has been proposed to explain the increase in the reduction potential, based upon changes in the Gly43-Ala44 peptide bond orientation and the distance to the [Fe(SCys)(4)] center in the Pyrococcus furiosus (Pf) Rd crystal structure compared to those of Gly43-Val44 in the Clostridium pasteurianum (Cp) Rd crystal structure. As an experimental test of this hypothesis, single-site Val44 <--> Ala44 exchange mutants, [V44A]Cp and [A44V]Pf Rds, have been cloned and expressed. Reduction potentials of these residue 44 variants and pertinent features of the X-ray crystal structure of [V44A]Cp Rd are reported. Relative to those of wild-type Cp and Pf Rds, the V44A mutation in Cp Rd results in an 86 mV increase in midpoint reduction potential and the [A44V] mutation in Pf Rd results in a 95 mV decrease in midpoint reduction potential, respectively. In the crystal structure of [V44A]Cp Rd, the peptide bond between residues 43 and 44 is approximately 0.3 A closer to the Fe center and the hydrogen bond distance between the residue 44 peptide nitrogen and the Cys42 gamma-sulfur decreases by 0.32 A compared to the analogous distances in the wild-type Cp Rd crystal structure. The results described herein support the prediction that the identity of residue 44 alone determines whether a Rd reduction potential of about -50 or 0 mV is observed.

Kinetics of the Superoxide Reductase Catalytic Cycle

“…The average k obs of ϳ240 s Ϫ1 coupled with the C13S 2Fe-SOR blue concentration of 200 M used in these experiments leads to an estimated second-order rate constant of 1.2 ϫ 10 6 M Ϫ1 s Ϫ1 for the reduced rubredoxin/C13S 2Fe-SOR blue electron transfer reaction. The fact that this value is about 4 times greater than the previously reported electron self-exchange rate constant for rubredoxin, ϳ3 ϫ 10 5 M Ϫ1 s Ϫ1 , can be qualitatively rationalized by the ϳ300 mV driving force for the reduced rubredoxin/ oxidized 2Fe-SOR electron transfer reaction (12,32) and the protein-surface accessible, solvent-exposed [Fe(His) 4 (Cys)] site of 2Fe-SOR (10). The limited amounts of C13S 2Fe-SOR available and the rapidity of the reaction prevented a more thorough kinetic analysis.…”

Eukaryotically Encoded and Chloroplast-located Rubredoxin Is Associated with Photosystem II

“…Redox Potential-To characterize the electrophysiological properties of the cryptomonad rubredoxin, we determined its redox potential under the same conditions as in Eidsness et al (22). Purified recombinant rubredoxin (8) was used to measure the redox potential.…”

“…The reason for this is a crucial amino acid at a conserved position (amino acid residue 44, referring to C. pasteurianum rubredoxin). Site-directed mutagenesis showed that changes from valine to the more polar alanine (and vice versa) correlate with the change of midpoint redox potential (22). Exclusively, the G. theta rubredoxin sequence has a serine at this position.…”

“Iron priming” guides folding of denatured aporubredoxins

“…Ultrapure urea and guanidine hydrochoride (GuHCl) were from Sigma. CpRds and PfRds were expressed and purified as the ironcontaining holoRds according to published procedures [29]. Unless otherwise indicated, all the experiments were carried out aerobically at room temperature in 50 mM tris(hydroxymethyl)aminomethane hydrochloride, pH 7.4 (hereafter referred to as buffer).…”

“…Stock solutions of 6-8 M urea or 6 M GuHCl were prepared in buffer. The concentrations of holoRds were determined using ε 492 = 8,850 M −1 cm −1 [23,29]. ApoCpRd and apoPfRd were prepared by treating the holoRds with 10 or 25% trichloroacetic acid, respectively, in the presence of 5 mM 2-mercaptoethanol at 0 °C.…”