The molecular determinants of the increased reduction potential of the rubredoxin domain of rubrerythrin relative to rubredoxin

Abstract

Based on the crystal structures, three possible sequence determinants have been suggested as the cause of a 285 mV increase in reduction potential of the rubredoxin domain of rubrerythrin over rubredoxin by modulating the polar environment around the redox site. Here, electrostatic calculations of crystal structures of rubredoxin and rubrerythrin and molecular dynamics simulations of rubredoxin wild-type and mutants are used to elucidate the contributions to the increased reduction potential. Asn(160) and His(179) in rubrerythrin versus valines in rubredoxins are predicted to be the major contributors, as the polar side chains contribute significantly to the electrostatic potential in the redox site region. The mutant simulations show both side chains rotating on a nanosecond timescale between two conformations with different electrostatic contributions. Reduction also causes a change in the reduction energy that is consistent with a linear response due to the interesting mechanism of shifting the relative populations of the two conformations. In addition to this, a simulation of a triple mutant indicates the side-chain rotations are approximately anticorrelated so whereas one is in the high potential conformation, the other is in the low potential conformation. However, Ala(176) in rubrerythrin versus a leucine in rubredoxin is not predicted to be a large contributor, because the solvent accessibility increases only slightly in mutant simulations and because it is buried in the interface of the rubrerythrin homodimer.

Figure 1

Ribbon diagram depicting the Dv (1LKM) rubrerythrin homodimer in blue, with the metal atoms for the diiron site and the Fe(S)₄ site shown as gray van der Waals spheres.

Figure 2

Superposition of the DvRr in oxidized (1LKM) and reduced (1LKO) states (blue and ice blue) and CpRd in oxidized (1FHH) and reduced (1FHM) states (red and pink) near the Fe(S)₄ metal center (white and yellow), aligned on the Fe(S)₄ center.

Figure 3

Licorice model depicting the backbone and side-chain relation interaction with the Fe(S)₄ center (white and yellow) of the average MD structures of (a) the oxidized and reduced V8N CpRd (green and cyan), the oxidized and reduced wt CpRd (red and pink), (b) the oxidized and reduced V44H CpRd (blue and ice blue), and the oxidized and reduced wt CpRd (red and pink). Also shown is the DvRr (1LKM) crystal structure (ochre). The alignment is based on the Fe(S)₄ center.