Modulation of the redox potential of the [Fe(SCys)4] site in rubredoxin by the orientation of a peptide dipole

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 similar to 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 er;pressed. 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 similar to 0.3 Angstrom 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 Angstrom 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.