X-ray absorption spectroscopic characterization of the diferric-peroxo intermediate of human deoxyhypusine hydroxylase in the presence of its substrate eIF5a

Abstract

Human deoxyhypusine hydroxylase (hDOHH) is an enzyme that is involved in the critical post-translational modification of the eukaryotic translation initiation factor 5A (eIF5A). Following the conversion of a lysine residue on eIF5A to deoxyhypusine (Dhp) by deoxyhypusine synthase, hDOHH hydroxylates Dhp to yield the unusual amino acid residue hypusine (Hpu), a modification that is essential for eIF5A to promote peptide synthesis at the ribosome, among other functions. Purification of hDOHH overexpressed in E. coli affords enzyme that is blue in color, a feature that has been associated with the presence of a peroxo-bridged diiron(III) active site. To gain further insight into the nature of the diiron site and how it may change as hDOHH goes through the catalytic cycle, we have conducted X-ray absorption spectroscopic studies of hDOHH on five samples that represent different species along its reaction pathway. Structural analysis of each species has been carried out, starting with the reduced diferrous state, proceeding through its O2 adduct, and ending with a diferric decay product. Our results show that the Fe•••Fe distances found for the five samples fall within a narrow range of 3.4–3.5 Å, suggesting that hDOHH has a fairly constrained active site. This pattern differs significantly from what has been associated with canonical dioxygen activating nonheme diiron enzymes such as soluble methane monooxygenase and Class 1A ribonucleotide reductases, for which the Fe•••Fe distance can change by as much as 1 Å during the redox cycle. These results suggest that the O2 activation mechanism for hDOHH deviates somewhat from that associated with the canonical nonheme diiron enzymes, opening the door to new mechanistic possibilities for this intriguing family of enzymes.

A Carboxylate Shift Regulates Dioxygen Activation by the Diiron Nonheme β-Hydroxylase CmlA upon Binding of a Substrate-Loaded Nonribosomal Peptide Synthetase

“…Extra carbon scatterers at 3.56 Å also improve the fit, and are consistent with assignment to the C β atom of an N δ -bound histidine ligand (Table S3, fit 22 vs 23). 35 In addition, the EXAFS fit does not include the carbon scatterer at ~2.6 Å that is associated with the terminal bidentate carboxylate found in the crystal structure of WT R and observed in its EXAFS fit. This result would be consistent with this carboxylate no longer coordinating in a bidentate mode upon binding of untethered CmlP AT to WT R .…”

“…35 The relative invariance of the cluster structure was proposed to be a consequence of the unique HEAT-repeat protein fold in DOHH that allowed for rigid cross-domain binding of the 4-His-2-carboxylate ligand framework to the Fe centers. It could be argued that the unusual metallo-β-lactamase fold of CmlA may also innately impart rigidity to the diiron cluster, but our data does not provide us clear insight into the issue.…”

Structure/function correlations over binuclear non-heme iron active sites

“…The Mn(III)/ Mn(IV) center can be probed by MCD and the Fe(III) center by NRVS to correlate with the binuclear non-heme Fe enzymes [93]. Finally, a number of new biferrous enzyme classes are emerging (deoxyhypusine hydroxylase (DOHH) [94, 131], urease [95], CmlI [132], etc.) that are accessible to the VTVH MCD/DFT approach presented above to extend Table 1 and develop additional structure/function correlations.…”

Peroxide Activation for Electrophilic Reactivity by the Binuclear Non-heme Iron Enzyme AurF

“…These spectral changes have been attributed to the presence of an additional μ -hydroxo bridge. 18,27 Recently, the crystal structure of toluene 4-monooxygenase (T4MO) has been obtained with O 2 and substrate bound, and based on Fe–O distances, an Fe(II)Fe(III)-superoxide species rather than a peroxo biferric species has been suggested as the active intermediate. 28 …”