A Single Outer-Sphere Mutation Stabilizes apo-Mn Superoxide Dismutase by 35 °C and Disfavors Mn Binding

Abstract

The catalytic active site of Mn-specific SOD (MnSOD) is organized around a redox-active Mn ion. The most highly-conserved difference between MnSODs and the homologous FeSODs is the origin of a Gln in the second coordination sphere. In MnSODs it derives from the C-terminal domain whereas in FeSODs it derives from the N-terminal domain, yet its side chain occupies almost superimposable positions in the two types of SODs’ active sites. Mutation of this Gln69 to Glu in E. coli FeSOD increased the Fe3+/2+ reduction midpoint potential by > 0.6 V without disrupting the structure or Fe binding [E. Yikilmaz, D. W. Rodgers and A.-F. Miller (2006) Biochemistry 45(4) 1151–1161]. We now describe the analogous Q146E mutant of MnSOD, explaining its low Mn content in terms increased stability of the apo-Mn protein. In 0.8 M guanidinium HCl, the Q146E-apoMnSOD displays an apparent melting midpoint temperature (Tm) 35 °C higher that of WT-apoMnSOD, whereas the Tm of WT-holoMnSOD is only 20 °C higher than that of WT-apoMnSOD. In contrast, the Tm attributed to Q146E-holoMnSOD is 40 °C lower than that of Q146E-apoMnSOD. Thus our data refute the notion that the WT residues optimize structural stability of the protein, being instead consistent with conservation on the basis of enzyme function and therefore ability to bind metal ion. We propose that the WT-MnSOD protein conserves a destabilizing amino acid at position 146 as part of a strategy for favoring metal ion binding.

Interchangeable utilization of metals: New perspectives on the impacts of metal ions employed in ancient and extant biomolecules

“…Catalytically active sites of both SODs contain three histidines and one aspartic acid that bind the metal ion co-factor (Figure 4A, left and middle panels). In addition, the Fe and Mn ions also bind to a solvent molecule (water or hydroxide) that is engaged in hydrogen bonds formation and, together with histidines and aspartic acid, participates in the coordination of either cation in an unusual distorted trigonal bipyramidal geometry around the metal center, as depicted in Figure 2B (128)(129)(130). Despite this remarkable similarity, most Fe-SOD and Mn-SOD enzymes are only functional when bound to their cognate metal ion (Table 1), illustrating their high specificity to metal co-factor (131)(132)(133).…”

“…4 A , left and middle panels ). In addition, the Fe and Mn ions also bind to a solvent molecule (water or hydroxide) that is engaged in the formation of hydrogen bonds and, together with histidines and aspartic acid, participates in the coordination of either cation in an unusual distorted trigonal bipyramidal geometry around the metal center, as depicted in Figure 2 B ( 126 , 127 , 128 ).

Figure 4 Structural properties of Mn-specific, Fe-specific, and cambialistic Mn/Fe-SODs.

…”

Structural Basis for the Effects of Phenylalanine on Tuning the Reduction Potential of Type 1 Copper in Azurin

“…In comparison to studying PCS effects, probing the SCS is often more challenging, as the latter typically induces more subtle electronic and structural perturbations and thus requires more careful experimental design and detailed characterization. Toward this goal, several studies have contributed to the advance of this field that include not only T1Cu proteins − but also other redox-active proteins such as those containing purple Cu A center, − iron and manganese superoxide dismutase, − the heme-Cu B active site cytochrome c oxidase , and Fe-S proteins. ,− In previous publications, we have shown that E °′ of T1Cu azurin (Az) can be fine-tuned across a wide range of E °′ under physiological conditions, i.e., from −1 to +1 V vs standard hydrogen electrode (SHE) (all E °′ stated hereafter are vs SHE) by altering the hydrophobicity, hydrogen bond interactions, and the coordinated metals (Cu vs Ni) in the SCS of the T1Cu center. , The introduction of phenylalanine (Phe) to the SCS has been critical in accomplishing this tuning, as it allows the hydrophobicity of the T1Cu to be modulated. For example, the introduction of M44F and G116F to the N47S/F114N/M121L-Az variant resulted in an increase of E °′ from ∼690 to 970 ± 20 mV at pH 5.0, the highest E °′ of any engineered copper protein to date. , Despite these successes, the structural basis for such hydrophobicity-based redox tuning using Phe has not been elucidated.…”