Assembly of a heterodinuclear Mn/Fe cofactor is coupled to tyrosine–valine ether cross-link formation in the R2-like ligand-binding oxidase

Abstract

R2-like ligand-binding oxidases (R2lox) assemble a heterodinuclear Mn/Fe cofactor which performs reductive dioxygen (O 2 ) activation, catalyzes formation of a tyrosine–valine ether cross-link in the protein scaffold, and binds a fatty acid in a putative substrate channel. We have previously shown that the N-terminal metal binding site 1 is unspecific for manganese or iron in the absence of O 2 , but prefers manganese in the presence of O 2 , whereas the C-terminal site 2 is specific for iron. Here, we analyze the effects of amino acid exchanges in the cofactor environment on cofactor assembly and metalation specificity using X-ray crystallography, X-ray absorption spectroscopy, and metal quantification. We find that exchange of either the cross-linking tyrosine or the valine, regardless of whether the mutation still allows cross-link formation or not, results in unspecific manganese or iron binding at site 1 both in the absence or presence of O 2 , while site 2 still prefers iron as in the wild-type. In contrast, a mutation that blocks binding of the fatty acid does not affect the metal specificity of either site under anoxic or aerobic conditions, and cross-link formation is still observed. All variants assemble a dinuclear trivalent metal cofactor in the aerobic resting state, independently of cross-link formation. These findings imply that the cross-link residues are required to achieve the preference for manganese in site 1 in the presence of O 2 . The metalation specificity, therefore, appears to be established during the redox reactions leading to cross-link formation. Electronic supplementary material The online version of this article (10.1007/s00775-019-01639-4) contains supplementary material, which is available to authorized users.

Location-specific quantification of protein-bound metal ions by X-ray anomalous dispersion: Q-XAD

“…Q-XAD is the only metal-quantification method that provides location-specific information. Our results show that Q-XAD is reliable (Griese et al, 2013(Griese et al, , 2019Kutin et al, 2016). We have previously shown that the quantification results agree with those obtained by other methods such as EPR and Mö ssbauer spectroscopy as well as TXRF (Kutin et al, 2016).…”

“…a reduced versus an oxidized state of a redoxactive metal cluster), we recommend choosing an X-ray energy slightly above the absorption edge in order to be certain that the energy is never below the edge. In our experiments, we used the theoretical edge plus 50 eV (Griese et al, 2013(Griese et al, , 2019Kutin et al, 2016).…”

“…Hence, site 1 does not behave according to expectation: the protein scaffold defies the IW series. Dissection of the cofactor-assembly mechanism of R2lox indicated that both thermodynamic and kinetic preferences contribute to the observed metal selectivity, although the precise specificity determinants remain elusive (Griese et al, 2013(Griese et al, , 2019Kutin et al, 2016). Here, we investigated the effect of competition by transition metals higher in the IW series, i.e.…”

Chemical flexibility of heterobimetallic Mn/Fe cofactors: R2lox and R2c proteins

“…Another second-sphere substitution roughly opposite to G68 in the ligand-binding tunnel, A171F (see Figure 2F), demonstrates that the different effects of the G68L and G68F substitutions are not a consequence of the absence or presence of the fatty acid. The A171F substitution completely blocks ligand binding to the active site, but has no discernible effect on cofactor assembly or cross-link formation (38), indicating that the phenotype of G68L-R2lox is caused specifically by this mutation. We further characterized the interesting effects of the G68L mutation using EPR and X-ray spectroscopy as well as mass spectrometry.…”

“…Thus, although G68L-R2lox efficiently assembles Mn/Fe cofactors in solution which reduce O2, the leucine substitution does impair the catalytic process coupled to cofactor assembly, i.e. cross-link formation (38). It is not possible to determine the total amount of cross-link per protein, as only relative amounts of the same peptide across different samples can be determined.…”