A combined experimental and computational study of ligand-controlled Chan-Lam coupling of sulfenamides.

The unique features of the sulfenamides' S(II)-N bond lead to interesting stereochemical properties and significant industrial functions. Here we present a chemoselective Chan-Lam coupling of sulfenamides to prepare N-arylated sulfenamides. A tridentate pybox ligand governs the chemoselectivity favoring C-N bond formation, and overrides the competitive C-S bond formation by preventing the S,N-bis-chelation of sulfenamides to copper center. The Cu(II)-derived resting state of catalyst is captured by UV-Vis spectra and EPR technique, and the key intermediate is confirmed by the EPR isotope response using 15N-labeled sulfenamide. A computational mechanistic study reveals that N-arylation is both kinetically and thermodynamically favorable, with deprotonation of the sulfenamide nitrogen atom occurring prior to reductive elimination. The origin of ligand-controlled chemoselectivity is explored, with the interaction between the pybox ligand and the sulfenamide substrate controlling the energy of the S-arylation and the corresponding product distribution, in agreement with the EPR studies and kinetic results.

Biosynthesis of GMGT lipids by a radical SAM enzyme associated with anaerobic archaea and oxygen-deficient environments.

Archaea possess characteristic membrane-spanning lipids that are thought to contribute to the adaptation to extreme environments. However, the biosynthesis of these lipids is poorly understood. Here, we identify a radical S-adenosyl-L-methionine (SAM) enzyme that synthesizes glycerol monoalkyl glycerol tetraethers (GMGTs). The enzyme, which we name GMGT synthase (Gms), catalyzes the formation of a C(sp3)-C(sp3) linkage between the two isoprenoid chains of glycerol dialkyl glycerol tetraethers (GDGTs). This conclusion is supported by heterologous expression of gene gms from a GMGT-producing species in a methanogen, as well as demonstration of in vitro activity using purified Gms enzyme. Additionally, we show that genes encoding putative Gms homologs are present in obligate anaerobic archaea and in metagenomes obtained from oxygen-deficient environments, and appear to be absent in metagenomes from oxic settings.

An explainable multiscale LSTM model with wavelet transform and layer-wise relevance propagation for daily streamflow forecasting.

Developing an accurate and reliable daily streamflow forecasting model is important for facilitating the efficient resource planning and management of hydrological systems. In this study, an explainable multiscale long short-term memory (XM-LSTM) model is proposed for effective daily streamflow forecasting by integrating the à trous wavelet transform (ATWT) for decomposing data, the Boruta algorithm for identifying model inputs, and the layer-wise relevance propagation (LRP) for explaining the prediction results. The proposed XM-LSTM is tested by performing multi-step-ahead forecasting of daily streamflow at four stations in the middle and lower reaches of the Yangtze River basin and compared with the X-LSTM. The X-LSTM is formed by coupling the long short-term memory (LSTM) with the LRP. For comparison, the inputs of these two models are identified by the Boruta selection algorithm. The results show that all models exhibit good ability to forecast daily streamflow, however, the prediction performance decreases as the lead time increases. The XM-LSTM provides a better forecasting performance than the X-LSTM, suggesting the ability of the ATWT to improve the LSTM for daily streamflow forecasting. Moreover, the correlation scores analysis by the LRP shows that the ATWT can extract useful information that influences the daily streamflow from the raw predictors, and the water level has the most significant contribution to streamflow prediction. Accordingly, the XM-LSTM model can be viewed as a potentially useful approach for increasing the accuracy and explainability of streamflow forecasting.

Radical SAM Enzyme PylB Generates a Lysyl Radical Intermediate in the Biosynthesis of Pyrrolysine by Using SAM as a Cofactor.

Pyrrolysine, the 22nd amino acid encoded by the natural genetic code, is essential for methanogenic archaea to catabolize methylamines into methane. The structure of pyrrolysine consists of a methylated pyrroline carboxylate that is linked to the ε-amino group of the l-lysine via an amide bond. The biosynthesis of pyrrolysine requires three enzymes: PylB, PylC, and PylD. PylB is a radical S-adenosyl-l-methionine (SAM) enzyme and catalyzes the first biosynthetic step, the isomerization of l-lysine into methylornithine. PylC catalyzes an ATP-dependent ligation of methylornithine and a second l-lysine to form l-lysine-Nε-methylornithine. The last biosynthetic step is catalyzed by PylD via oxidation of the PylC product to form pyrrolysine. While enzymatic reactions of PylC and PylD have been well characterized by X-ray crystallography and in vitro studies, mechanistic understanding of PylB is still relatively limited. Here, we report the first in vitro activity of PylB to form methylornithine via the isomerization of l-lysine. We also identify a lysyl C4 radical intermediate that is trapped, with its electronic structure and geometric structure well characterized by EPR and ENDOR spectroscopy. In addition, we demonstrate that SAM functions as a catalytic cofactor in PylB catalysis rather than canonically as a cosubstrate. This work provides detailed mechanistic evidence for elucidating the carbon backbone rearrangement reaction catalyzed by PylB during the biosynthesis of pyrrolysine.

An Isolable Radical Anion Featuring a 2-Center-3-Electron π-Bond without a Clearly Defined σ-Bond.

Featuring an extra electron in the π* antibonding orbital, species with a 2-center-3-electron (2c3e) π bond without an underlying σ bond are scarcely known. Herein, we report the synthesis, isolation and characterization of a radical anion salt [K(18-C-6)]+ {[(HCNDipp)2 Si]2 P2 }⋅- (i.e. [K(18-C-6)]+ 3⋅- ) (18-C-6=18-crown-6, Dipp=2,6-diisopropylphenyl), in which 3⋅- features a perfectly planar Si2 P2 four-membered ring. This species represents the first example of a Si- and P-containing analog of a bicyclo[1.1.0]butane radical anion. The unusual bonding motif of 3⋅- was thoroughly investigated via X-ray diffraction crystallography, electron paramagnetic resonance spectroscopy (EPR), and calculations by density functional theory (DFT), which collectively unveiled the existence of a 2c3e π bond between the bridgehead P atoms and no clearly defined supporting P-P σ bond.

Characterizing the Biosynthesis of the [Fe(II)(CN)(CO)2(cysteinate)]- Organometallic Product of the Radical-SAM Enzyme HydG by EPR and Mössbauer Spectroscopy.

[FeFe]-hydrogenases employ a catalytic H-cluster, consisting of a [4Fe-4S]H cluster linked to a [2Fe]H subcluster with CO, CN- ligands, and an azadithiolate bridge, which mediates the rapid redox interconversion of H+ and H2. In the biosynthesis of this H-cluster active site, the radical S-adenosyl-l-methionine (radical SAM, RS) enzyme HydG plays the crucial role of generating an organometallic [Fe(II)(CN)(CO)2(cysteinate)]- product that is en route to forming the H-cluster. Here, we report direct observation of this diamagnetic organometallic Fe(II) complex through Mössbauer spectroscopy, revealing an isomer shift of δ = 0.10 mm s-1 and quadrupole splitting of ΔEQ = 0.66 mm s-1. These Mössbauer values are a change from the starting values of δ = 1.15 mm s-1 and ΔEQ = 3.23 mm s-1 for the ferrous "dangler" Fe in HydG. These values of the observed product complex B are in good agreement with Mössbauer parameters for the low-spin Fe2+ ions in synthetic analogues, such as 57Fe Syn-B, which we report here. These results highlight the essential role that HydG plays in converting a resting-state high-spin Fe(II) to a low-spin organometallic Fe(II) product that can be transferred to the downstream maturase enzymes.

Cross-coupling by a noncanonical mechanism involving the addition of aryl halide to Cu(II).

Copper complexes are widely used in the synthesis of fine chemicals and materials to catalyze couplings of heteroatom nucleophiles with aryl halides. We show that cross-couplings catalyzed by some of the most active catalysts occur by a mechanism not previously considered. Copper(II) [Cu(II)] complexes of oxalamide ligands catalyze Ullmann coupling to form the C-O bond in aryl ethers by concerted oxidative addition of an aryl halide to Cu(II) to form a high-valent species that is stabilized by radical character on the oxalamide ligand. This mechanism diverges from those involving Cu(I) and Cu(III) intermediates that have been posited for other Ullmann-type couplings. The stability of the Cu(II) state leads to high turnover numbers, >1000 for the coupling of phenoxide with aryl chloride electrophiles, as well as an ability to run the reactions in air.

Tuning the Type 1 Reduction Potential of Multicopper Oxidases: Uncoupling the Effects of Electrostatics and H-Bonding to Histidine Ligands.

In multicopper oxidases (MCOs), the type 1 (T1) Cu accepts electrons from the substrate and transfers these to the trinuclear Cu cluster (TNC) where O2 is reduced to H2O. The T1 potential in MCOs varies from 340 to 780 mV, a range not explained by the existing literature. This study focused on the ∼350 mV difference in potential of the T1 center in Fet3p and Trametes versicolor laccase (TvL) that have the same 2His1Cys ligand set. A range of spectroscopies performed on the oxidized and reduced T1 sites in these MCOs shows that they have equivalent geometric and electronic structures. However, the two His ligands of the T1 Cu in Fet3p are H-bonded to carboxylate residues, while in TvL they are H-bonded to noncharged groups. Electron spin echo envelope modulation spectroscopy shows that there are significant differences in the second-sphere H-bonding interactions in the two T1 centers. Redox titrations on type 2-depleted derivatives of Fet3p and its D409A and E185A variants reveal that the two carboxylates (D409 and E185) lower the T1 potential by 110 and 255-285 mV, respectively. Density functional theory calculations uncouple the effects of the charge of the carboxylates and their difference in H-bonding interactions with the His ligands on the T1 potential, indicating 90-150 mV for anionic charge and ∼100 mV for a strong H-bond. Finally, this study provides an explanation for the generally low potentials of metallooxidases relative to the wide range of potentials of the organic oxidases in terms of different oxidized states of their TNCs involved in catalytic turnover.

Characterizing SPASM/twitch Domain-Containing Radical SAM Enzymes by EPR Spectroscopy.

Owing to their importance, diversity and abundance of generated paramagnetic species, radical S-adenosylmethionine (rSAM) enzymes have become popular targets for electron paramagnetic resonance (EPR) spectroscopic studies. In contrast to prototypic single-domain and thus single-[4Fe-4S]-containing rSAM enzymes, there is a large subfamily of rSAM enzymes with multiple domains and one or two additional iron-sulfur cluster(s) called the SPASM/twitch domain-containing rSAM enzymes. EPR spectroscopy is a powerful tool that allows for the observation of the iron-sulfur clusters as well as potentially trappable paramagnetic reaction intermediates. Here, we review continuous-wave and pulse EPR spectroscopic studies of SPASM/twitch domain-containing rSAM enzymes. Among these enzymes, we will review in greater depth four well-studied enzymes, BtrN, MoaA, PqqE, and SuiB. Towards establishing a functional consensus of the additional architecture in these enzymes, we describe the commonalities between these enzymes as observed by EPR spectroscopy.

Thionitrite (SNO- ) and Perthionitrite (SSNO- ) are Simple Synthons for Nitrosylated Iron Sulfur Clusters.

S/N crosstalk species derived from the interconnected reactivity of H2 S and NO facilitate the transport of reactive sulfur and nitrogen species in signaling, transport, and regulatory processes. We report here that simple Fe2+ ions, such as those that are bioavailable in the labile iron pool (LIP), react with thionitrite (SNO- ) and perthionitrite (SSNO- ) to yield the dinitrosyl iron complex [Fe(NO)2 (S5 )]- . In the reaction of FeCl2 with SNO- we were able to isolate the unstable intermediate hydrosulfido mononitrosyl iron complex [FeCl2 (NO)(SH)]- , which was characterized by X-ray crystallography. We also show that [Fe(NO)2 (S5 )]- is a simple synthon for nitrosylated Fe-S clusters via its reduction with PPh3 to yield Roussin's Red Salt ([Fe2 S2 (NO)4 ]2- ), which highlights the role of S/N crosstalk species in the assembly of fundamental Fe-S motifs.

Proposed Mechanism for the Biosynthesis of the [FeFe] Hydrogenase H-Cluster: Central Roles for the Radical SAM Enzymes HydG and HydE.

Radical S-adenosylmethionine (radical SAM or rSAM) enzymes use their S-adenosylmethionine cofactor bound to a unique Fe of a [4Fe-4S] cluster to generate the "hot" 5'-deoxyadenosyl radical, which drives highly selective radical reactions via specific interactions with a given rSAM enzyme's substrate. This Perspective focuses on the two rSAM enzymes involved in the biosynthesis of the organometallic H-cluster of [FeFe] hydrogenases. We present here a detailed sequential model initiated by HydG, which lyses a tyrosine substrate via a 5'-deoxyadenosyl H atom abstraction from those amino acid's amino group, initially producing dehydroglycine and an oxidobenzyl radical. In this model, two successive radical cascade reactions lead ultimately to the formation of HydG's product, a mononuclear Fe organometallic complex: [Fe(II)(CN)(CO)2(cysteinate)]-, with the iron originating from a unique "dangler" Fe coordinated by a cysteine ligand providing a sulfur bridge to another [4Fe-4S] auxiliary cluster in the enzyme. In turn, in this model, [Fe(II)(CN)(CO)2(cysteinate)]- is the substrate for HydE, the second rSAM enzyme in the biosynthetic pathway, which activates this mononuclear organometallic unit for dimerization, forming a [Fe2S2(CO)4(CN)2] precursor to the [2Fe] H component of the H-cluster, requiring only the completion of the bridging azadithiolate (SCH2NHCH2S) ligand. This model is built upon a foundation of data that incorporates cell-free synthesis, isotope sensitive spectroscopies, and the selective use of synthetic complexes substituting for intermediates in the enzymatic "assembly line". We discuss controversies pertaining to this model and some remaining open issues to be addressed by future work.

Organometallic Fe2(µ-SH)2(CO)4(CN)2 Cluster Allows the Biosynthesis of the [FeFe]-Hydrogenase with Only the HydF Maturase.

The biosynthesis of the active site of the [FeFe]-hydrogenases (HydA1), the H-cluster, is of interest because these enzymes are highly efficient catalysts for the oxidation and production of H2. The biosynthesis of the [2Fe]H subcluster of the H-cluster proceeds from simple precursors, which are processed by three maturases: HydG, HydE, and HydF. Previous studies established that HydG produces an Fe(CO)2(CN) adduct of cysteine, which is the substrate for HydE. In this work, we show that by using the synthetic cluster [Fe2(µ-SH)2(CN)2(CO)4]2- active HydA1 can be biosynthesized without maturases HydG and HydE.

Biosynthesis of fluopsin C, a copper-containing antibiotic from Pseudomonas aeruginosa.

Metal-binding natural products contribute to metal acquisition and bacterial virulence, but their roles in metal stress response are underexplored. We show that a five-enzyme pathway in Pseudomonas aeruginosa synthesizes a small-molecule copper complex, fluopsin C, in response to elevated copper concentrations. Fluopsin C is a broad-spectrum antibiotic that contains a copper ion chelated by two minimal thiohydroxamates. Biosynthesis of the thiohydroxamate begins with cysteine and requires two lyases, two iron-dependent enzymes, and a methyltransferase. The iron-dependent enzymes remove the carboxyl group and the α carbon from cysteine through decarboxylation, N-hydroxylation, and methylene excision. Conservation of the pathway in P. aeruginosa and other bacteria suggests a common role for fluopsin C in the copper stress response, which involves fusing copper into an antibiotic against other microbes.

Metallo-inhibition of Mnx, a bacterial manganese multicopper oxidase complex.

The manganese oxidase complex, Mnx, from Bacillus sp. PL-12 contains a multicopper oxidase (MCO) and oxidizes dissolved Mn(II) to form insoluble manganese oxide (MnO2) mineral. Previous kinetic and spectroscopic analyses have shown that the enzyme's mechanism proceeds through an activation step that facilitates formation of a series of binuclear Mn complexes in the oxidation states II, III, and IV on the path to MnO2 formation. We now demonstrate that the enzyme is inhibited by first-row transition metals in the order of the Irving-Williams series. Zn(II) strongly (Ki ~ 1.5 µM) inhibits both activation and turnover steps, as well as the rate of Mn(II) binding. The combined Zn(II) and Mn(II) concentration dependence establishes that the inhibition is non-competitive. This result is supported by electron paramagnetic resonance (EPR) spectroscopy, which reveals unaltered Mnx-bound Mn(II) EPR signals, both mono- and binuclear, in the presence of Zn(II). We infer that inhibitory metals bind at a site separate from the substrate sites and block the conformation change required to activate the enzyme, a case of allosteric inhibition. The likely biological role of this inhibitory site is discussed in the context of Bacillus spore physiology. While Cu(II) inhibits Mnx strongly, in accord with the Irving-Williams series, it increases Mnx activation at low concentrations, suggesting that weakly bound Cu, in addition to the four canonical MCO-Cu, may support enzyme activity, perhaps as an electron transfer agent.

Improving modeling of ecosystem gross primary productivity through re-optimizing temperature restrictions on photosynthesis.

The terrestrial ecosystem gross primary productivity (GPP) plays an important role in the global carbon cycle and ecosystem functions. However, the estimates of GPP still have large uncertainties due to insufficient understanding of the photosynthesis-temperature relationship and maximum light use efficiency (LUEmax). We used satellite-derived proxies of GPP to derive optimum, minimum, and maximum temperature for photosynthesis at the ecosystem scale, which was then used to construct a new temperature stress expression. This study improves the MODIS-based light use efficiency model through coupling the optimized LUEmax with the new proposed temperature stress expression. The new model (R2 = 0.81, RMSE = 17.8 gC m-2 (16 d)-1) performed better than the MODIS GPP products (R2 = 0.67, RMSE = 30.4 gC m-2 (16 d)-1), especially for evergreen broadleaf forests and croplands. The mean annual GPP over China is 5.7 ± 0.27 PgC, and the GPP significantly increased by 0.046 ± 0.006 PgC year-1 during 2001-2018. This study provides a potential method for future projections of terrestrial ecosystem functioning.

Crystal Structure of the [FeFe]-Hydrogenase Maturase HydE Bound to Complex-B.

[FeFe]-hydrogenases use a unique organometallic complex, termed the H cluster, to reversibly convert H2 into protons and low-potential electrons. It can be best described as a [Fe4S4] cluster coupled to a unique [2Fe]H center where the reaction actually takes place. The latter corresponds to two iron atoms, each of which is bound by one CN- ligand and one CO ligand. The two iron atoms are connected by a unique azadithiolate molecule (-S-CH2-NH-CH2-S-) and an additional bridging CO. This [2Fe]H center is built stepwise thanks to the well-orchestrated action of maturating enzymes that belong to the Hyd machinery. Among them, HydG converts l-tyrosine into CO and CN- to produce a unique l-cysteine-Fe(CO)2CN species termed complex-B. Very recently, HydE was shown to perform radical-based chemistry using synthetic complex-B as a substrate. Here we report the high-resolution crystal structure that establishes the identity of the complex-B-bound HydE. By triggering the reaction prior to crystallization, we trapped a new five-coordinate Fe species, supporting the proposal that HydE performs complex modifications of complex-B to produce a monomeric "SFe(CO)2CN" precursor to the [2Fe]H center. Substrate access, product release, and intermediate transfer are also discussed.

Trapping a cross-linked lysine-tryptophan radical in the catalytic cycle of the radical SAM enzyme SuiB.

The radical S-adenosylmethionine (rSAM) enzyme SuiB catalyzes the formation of an unusual carbon-carbon bond between the sidechains of lysine (Lys) and tryptophan (Trp) in the biosynthesis of a ribosomal peptide natural product. Prior work on SuiB has suggested that the Lys-Trp cross-link is formed via radical electrophilic aromatic substitution (rEAS), in which an auxiliary [4Fe-4S] cluster (AuxI), bound in the SPASM domain of SuiB, carries out an essential oxidation reaction during turnover. Despite the prevalence of auxiliary clusters in over 165,000 rSAM enzymes, direct evidence for their catalytic role has not been reported. Here, we have used electron paramagnetic resonance (EPR) spectroscopy to dissect the SuiB mechanism. Our studies reveal substrate-dependent redox potential tuning of the AuxI cluster, constraining it to the oxidized [4Fe-4S]2+ state, which is active in catalysis. We further report the trapping and characterization of an unprecedented cross-linked Lys-Trp radical (Lys-Trp•) in addition to the organometallic Ω intermediate, providing compelling support for the proposed rEAS mechanism. Finally, we observe oxidation of the Lys-Trp• intermediate by the redox-tuned [4Fe-4S]2+ AuxI cluster by EPR spectroscopy. Our findings provide direct evidence for a role of a SPASM domain auxiliary cluster and consolidate rEAS as a mechanistic paradigm for rSAM enzyme-catalyzed carbon-carbon bond-forming reactions.

Structural Properties and Catalytic Implications of the SPASM Domain Iron-Sulfur Clusters in Methylorubrum extorquens PqqE.

Understanding the relationship between the metallocofactor and its protein environment is the key to uncovering the mechanism of metalloenzymes. PqqE, a radical S-adenosylmethionine enzyme in pyrroloquinoline quinone (PQQ) biosynthesis, contains three iron-sulfur cluster binding sites. Two auxiliary iron-sulfur cluster binding sites, designated as AuxI and AuxII, use distinctive ligands compared to other proteins in the family while their functions remain unclear. Here, we investigate the electronic properties of these iron-sulfur clusters and compare the catalytic efficiency of wild-type (WT) Methylorubrum extorquens AM1 PqqE to a range of mutated constructs. Using native mass spectrometry, protein film electrochemistry, and electron paramagnetic resonance spectroscopy, we confirm the previously proposed incorporation of a mixture of [2Fe-2S] and [4Fe-4S] clusters at the AuxI site and are able to assign redox potentials to each of the three iron-sulfur clusters. Significantly, a conservative mutation at AuxI, C268H, shown to selectively incorporate a [4Fe-4S] cluster, catalyzes an enhancement of uncoupled S-adenosylmethionine cleavage relative to WT, together with the elimination of detectable peptide cross-linked product. While a [4Fe-4S] cluster can be tolerated at the AuxI site, the aggregate findings suggest a functional [2Fe-2S] configuration within the AuxI site. PqqE variants with nondestructive ligand replacements at AuxII also show that the reduction potential at this site can be manipulated by changing the electronegativity of the unique aspartate ligand. A number of novel mechanistic features are proposed based on the kinetic and spectroscopic data. Additionally, bioinformatic analyses suggest that the unique ligand environment of PqqE may be relevant to its role in PQQ biosynthesis within an oxygen-dependent biosynthetic pathway.

Both N-Terminal and C-Terminal Histidine Residues of the Prion Protein Are Essential for Copper Coordination and Neuroprotective Self-Regulation.

The cellular prion protein (PrPC) comprises two domains: a globular C-terminal domain and an unstructured N-terminal domain. Recently, copper has been observed to drive tertiary contact in PrPC, inducing a neuroprotective cis interaction that structurally links the protein's two domains. The location of this interaction on the C terminus overlaps with the sites of human pathogenic mutations and toxic antibody docking. Combined with recent evidence that the N terminus is a toxic effector regulated by the C terminus, there is an emerging consensus that this cis interaction serves a protective role, and that the disruption of this interaction by misfolded PrP oligomers may be a cause of toxicity in prion disease. We demonstrate here that two highly conserved histidines in the C-terminal domain of PrPC are essential for the protein's cis interaction, which helps to protect against neurotoxicity carried out by its N terminus. We show that simultaneous mutation of these histidines drastically weakens the cis interaction and enhances spontaneous cationic currents in cultured cells, the first C-terminal mutant to do so. Whereas previous studies suggested that Cu2+ coordination was localized solely to the protein's N-terminal domain, we find that both domains contribute equatorially coordinated histidine residue side-chains, resulting in a novel bridging interaction. We also find that extra N-terminal histidines in pathological familial mutations involving octarepeat expansions inhibit this interaction by sequestering copper from the C terminus. Our findings further establish a structural basis for PrPC's C-terminal regulation of its otherwise toxic N terminus.

Elucidation of a Copper Binding Site in Proinsulin C-peptide and Its Implications for Metal-Modulated Activity.

The connecting peptide (C-peptide) is a hormone with promising health benefits in ameliorating diabetes-related complications, yet mechanisms remain elusive. Emerging studies point to a possible dependence of peptide activity on bioavailable metals, particularly Cu(II) and Zn(II). However, little is known about the chemical nature of the interactions, hindering advances in its therapeutic applications. This work uncovers the Cu(II)-binding site in C-peptide that may be key to understanding its metal-dependent function. A combination of spectroscopic studies reveal that Cu(II) and Zn(II) bind to C-peptide at specific residues in the N-terminal region of the peptide and that Cu(II) is able to displace Zn(II) for C-peptide binding. The data point to a Cu(II)-binding site consisting of 1N3O square-planar coordination that is entropically driven. Furthermore, the entire random coil peptide sequence is needed for specific metal binding as mutations and truncations reshuffle the coordinating residues. These results expand our understanding of how metals influence hormone activity and facilitate the discovery and validation of both new and established paradigms in peptide biology.