Although metallocofactors are ubiquitous in enzyme catalysis, how metal binding specificity arises remains poorly understood, especially in the case of metals with similar primary ligand preferences such as manganese and iron. The biochemical selection of manganese over iron presents a particularly intricate problem because manganese is generally present in cells at a lower concentration than iron, while also having a lower predicted complex stability according to the Irving-Williams series (Mn II < Fe II < Ni II < Co II < Cu II > Zn II ). Here we show that a heterodinuclear Mn/Fe cofactor with the same primary protein ligands in both metal sites self-assembles from Mn II and Fe II in vitro, thus diverging from the Irving-Williams series without requiring auxiliary factors such as metallochaperones. Crystallographic, spectroscopic, and computational data demonstrate that one of the two metal sites preferentially binds Fe II over Mn II as expected, whereas the other site is nonspecific, binding equal amounts of both metals in the absence of oxygen. Oxygen exposure results in further accumulation of the Mn/Fe cofactor, indicating that cofactor assembly is at least a twostep process governed by both the intrinsic metal specificity of the protein scaffold and additional effects exerted during oxygen binding or activation. We further show that the mixed-metal cofactor catalyzes a two-electron oxidation of the protein scaffold, yielding a tyrosine-valine ether cross-link. Theoretical modeling of the reaction by density functional theory suggests a multistep mechanism including a valyl radical intermediate.H alf of all enzymes are estimated to contain metallocofactors (1). An important subset uses transition metal ions to perform key redox reactions such as oxygen activation. The diiron cofactor of the ferritin-like superfamily of proteins is particularly versatile (2). While ferritin itself simply oxidizes and sequesters iron (3), in other family members the diiron center acts as a transient one-or two-electron oxidant. In the R2 subunits of class I ribonucleotide reductases (RNRs) it generates a redoxactive tyrosyl radical (4, 5), whereas in the bacterial multicomponent monooxygenases (BMMs) it catalyzes the hydroxylation of a variety of hydrocarbons (6). For four decades it was assumed that all ferritin superfamily proteins contained diiron cofactors. However, in recent years new subfamilies harboring either a dimanganese or heterodinuclear Mn/Fe cofactor have been documented (7)(8)(9)(10)(11)(12)(13)(14). The Mn/Fe cofactor was discovered in class Ic RNR R2 subunits, where its Mn IV /Fe III state functionally replaces the diiron-tyrosyl radical cofactor of class Ia R2s (9, 10). After a long controversy, class Ib R2 proteins were shown to use a dimanganese cofactor in the same scaffold (7,8). These recent developments highlight the complexity of correctly identifying the metals that make up native metallocofactors. While the metal preferences of some primary coordination motifs are well known and distinct, others ar...

We present a liquid chromatography-mass spectrometry (LC-MS)-based method permitting unbiased (gene prediction-independent) genome-wide discovery of protein-coding loci in higher eukaryotes. Using high-resolution isoelectric focusing (HiRIEF) at the peptide level in the 3.7-5.0 pH range and accurate peptide isoelectric point (pI) prediction, we probed the six-reading-frame translation of the human and mouse genomes and identified 98 and 52 previously undiscovered protein-coding loci, respectively. The method also enabled deep proteome coverage, identifying 13,078 human and 10,637 mouse proteins.

Proteogenomics enable the discovery of novel peptides (from unannotated genomic protein-coding loci) and single amino acid variant peptides (derived from single-nucleotide polymorphisms and mutations). Increasing the reliability of these identifications is crucial to ensure their usefulness for genome annotation and potential application as neoantigens in cancer immunotherapy. We here present integrated proteogenomics analysis workflow (IPAW), which combines peptide discovery, curation, and validation. IPAW includes the SpectrumAI tool for automated inspection of MS/MS spectra, eliminating false identifications of single-residue substitution peptides. We employ IPAW to analyze two proteomics data sets acquired from A431 cells and five normal human tissues using extended (pH range, 3–10) high-resolution isoelectric focusing (HiRIEF) pre-fractionation and TMT-based peptide quantitation. The IPAW results provide evidence for the translation of pseudogenes, lncRNAs, short ORFs, alternative ORFs, N-terminal extensions, and intronic sequences. Moreover, our quantitative analysis indicates that protein production from certain pseudogenes and lncRNAs is tissue specific.