The Coordination Chemistry of Heterobimetallic Proteins

  Approximately one third of all proteins coordinate metal ions, and these metallocofactors must be selectively assembled in vivo for the protein to perform its intended biological function. In many cases, metallocofactors are assembled from a labile metal pool, a collection of metal ions in the cytoplasm coordinated by water and other small molecules., The thermodynamics of cofactor assembly are determined by the stability of the metallated protein relative to the complexes found in the labile pool. We are interested in using synthetic model complexes to understand how proteins exploit differences in metal ion coordination chemistry to selectively assemble heterobimetallic cofactors.


  Class I ribonucleotide reductases (RNR) catalyze the conversion of RNA to DNA. The enzyme complex contains an R2 subunit that generates a radical using O2 and a dinuclear metallocofactor, usually Fe/Fe or Mn/Mn. The recently identified Ic subclass of this enzyme (R2c) contains an unusual Mn/Fe cofactor that makes the enzyme more resistant to oxidative stress than its homometallic counterparts, but also requires the protein to discriminate between two very similar metals. Other Mn/Fe cofactors were subsequently identified in a family of ligand binding oxidases (R2lox) homologous with R2c.

 

Dinucleating ligand F-HXTA used to model R2c/R2lox metal binding sites (Left). Displacement ellipsoid plot of the complex anion [MnII2(F-HXTA)(H2O)4]-. (Right) A crystal structure of the analogous iron(II) complex was also obtained.

Equilibrium binding of Fe(II) and Mn(II) to Ct R2c has been reported by Dassama et al. and a strong preference for the correct {MnII(1)FeII(2)} cofactor was observed. The explanation for this selectivity has been discussed extensively, but what is particularly intriguing is the mechanism by which site 1 subverts the usual preference for FeII over MnII. A comparison of binding sites in a series of RNR R2 class Ia, b and c proteins by Dassama et al. suggests that MnII selectivity may result from the presence of exogenous water ligands that allow a more flexible coordination geometry. Similar arguments have been made for small-molecule coordination complexes that prefer MnII to FeII. Although there are probably many contributing factors to selective cofactor assembly in R2c and R2lox proteins, our results suggest a new possibility should be considered: Binding of MnII in site 1 may be affected by the presence of FeII in site 2, not just through the rearrangement of residues in the adjacent site, but also through an intrinsic favorable interaction between the metal ions in a {MnII FeII} cluster.


    Kerber, W. D.; Goheen, J. T.; Perez, K. A.; Siegler, M. A.; “Enhanced Stability of the FeII/MnII State in a Synthetic Model of Heterobimetallic Cofactor AssemblyInorg. Chem. 2016, 55, 848.

We have used the dinucleating ligand F-HXTA as a model of R2c/R2lox active sites in order to investigate the thermodynamics of Fe(II)/Mn(II) discrimination. Homometallic diiron(II) and dimanganese(II) F-HXTA complexes were prepared and characterized in the solid state by single crystal X-ray diffraction, and in solution (for iron) by 1H and 19F NMR. Metal ion exchange was shown to be facile in solution and 19F-NMR is a convenient tool to measure concentrations of paramagnetic M2(F-HXTA) species.

Concentrations of M2(F-HXTA) were measured as a function of metal ion ratio and from these data the equilibrium constants for F-HXTA metal ion exchange were determined (KT, K1, K2). The complete replacement of MnII with FeII is described by KT and was found to be 182 ± 13. K1 and K2 describe stepwise replacement reactions (where KT = K1 × K2) and these were found to be 20.1 ± 1.3 and 9.1 ± 1.1 respectively.


The relationship between K1 and K2 determines the relative stability of the heterobimetallic complex [FeMn(F-HXTA)]-. If the two metal binding sites in F-HXTA were completely independent from one another, each Mn/Fe substitution would be isoenergetic and the stepwise replacement constants would be equal (K1 = K2 = √ KT). If this were observed, the preference for Fe could be explained by the same arguments used for mononuclear complexes (ionic radii, dn configurations, HSAB theory). Although these factors are certainly still important, the observation that K1 > K2 indicates there is an additional favorable interaction specific to the heterobimetallic state.