The small, cysteine-rich metallothionein family of proteins is currently considered to play a critical role in the provision of metals to metalloenzymes. However, there is limited information available on the mechanisms of these fundamentally important interactions. We report on the competitive zinc metalation of apocarbonic anhydrase in the presence of apometallothionein 1A using electrospray-ionization mass spectrometry. These experiments revealed the relative affinities of zinc to all species in solution. The carbonic anhydrase is shown to compete efficiently only against Zn5-7MT. The calculated equilibrium zinc binding constants of each of the 7 zinc metallothionein 1A species ranged from a high of (log(KF)) 12.5 to a low of 11.8. The 8 equilibrium constants connecting the 10 active species in competition for the zinc were modeled by fitting the KF values of the 8 competitive bimolecular reactions to the ESI-mass spectral data. These modeled K values are shown to be experimentally connected to the metalation efficiency of the carbonic anhydrase. The series of 7 metallothionein binding affinities for zinc highlight the buffering role of zinc metallothioneins that permit simultaneously zinc storage and zinc sensing. Finally, the significance of the multiple zinc binding affinities of zinc metallothionein is discussed in relation to zinc homeostasis.

Metallothioneins (MTs) are cysteine-rich, metal-binding proteins that are found throughout Nature. This ubiquity highlights their importance in essential metal regulation, heavy metal detoxification and cellular redox chemistry. Missing from the current description of MT function is the underlying mechanism by which MTs achieve their proposed biological functions. To date, there have been conflicting reports on the mechanism of metal binding and the structures of the metal binding intermediates formed during metalation of apoMTs. The form of the metal-bound intermediates dictates the metal sequestering and metal-donating properties of the protein. Through a detailed analysis of spectral data from electrospray ionization mass spectromeric and circular dichroism methods we report that Zn(ii) and Cd(ii) metalation of the human MT1a takes place through two distinct pathways. The first pathway involves formation of beaded structures with up to five metals bound terminally to the 20 cysteines of the protein via a noncooperative mechanism. The second pathway is dominated by the formation of the four-metal domain cluster structure M4SCYS11via a cooperative mechanism. We report that there are different pathway preferences for Zn(ii) and Cd(ii) metalation of apo-hMT1a. Cd(ii) binding follows the beaded pathway above pH 7.1 but beginning below pH 7.1 the clustered (Cd4Scys11) pathway begins to dominate. In contrast, Zn(ii) binding follows the terminal, "beaded", pathway at all physiologically relevant pH (pH ≥ 5.2) only following the clustered pathway below pH 5.1. The results presented here allow us to reconcile the conflicting reports concerning the presence of different metalation intermediates of MTs. The conflict regarding cooperative versus noncooperative binding mechanisms is also reconciled with the experimental results described here. These two metal-specific pathways and the presence of radically different intermediate structures provide insight into the multi-functional nature of MT: binding Zn(ii) terminally for donation to metalloenzymes and sequestering toxic Cd(ii) in a cluster structure.

Ga(III)protoporphyrin-IX (Ga-PP) has been proposed as a model for the key interporphyrin interactions in malaria pigment. Unlike the paramagnetic parent iron heme derivatives, Ga-PP is readily soluble in methanol (MeOH). We report optical, mass spectroscopic, and theoretical results for Ga-PP as well as its reactions with myoglobin. UV-visible absorption and MCD spectroscopy show that Ga-PP exhibits a typical spectrum for a main group metal: a Q-band at 539 nm and a B band at 406 nm when dissolved in MeOH. We also report optical data for Zn(II)protoporphyrin IX (Zn-PP) dissolved in MeOH, which exhibits a Q-band at 545 nm and a B band at 415 nm. ESI mass spectral data for Ga-PP dissolved in MeOH show the presence of predominantly monomers, with smaller fractions of dimers [(Ga-PP)(2)] and trimers. UV-visible and MCD absorption spectroscopy and ESI mass spectral data demonstrate the successful insertion of monomeric Ga-PP into apo-Mb. Ga-PP-Mb exhibits a B band at 417 nm and Q bands at 545 and 584 nm, which are all red-shifted from the free Ga-PP values. The calculated electronic structures and frontier molecular orbitals of Ga-PP, (Ga-PP)(2) and Zn-PP fit the previously reported trends in band energies and oscillator strengths as a function of molecular orbital energies. These new data can be applied to explain the experimentally observed optical spectroscopy. The observed Q-band energies are accounted for by calculated (HOMO-LUMO) gap of the frontier MOs, while the split in the two top occupied MOs accounts for the magnitude of the Q-band oscillator strength as well as the experimentally observed Q to B band energy separation. Although Ga-PP shares more spectroscopic properties with Zn-PP than it does with Fe(III)PPIX, the trivalent oxidation state allows this molecule to be used as a model for ferric hemes in heme proteins.

Staphylococcus aureus possesses a multitude of mechanisms by which it can obtain iron during growth under iron starvation conditions. It expresses an effective heme acquisition system (the iron-regulated surface determinant system), it produces two carboxylate-type siderophores staphyloferrin A and staphyloferrin B (SB), and it expresses transporters for many other siderophores that it does not synthesize. The ferric uptake regulator protein regulates expression of genes encoding all of these systems. Mechanisms of fine-tuning expression of ironregulated genes, beyond simple iron regulation via ferric uptake regulator, have not been uncovered in this organism. Here, we identify the ninth gene of the sbn operon, sbnI, as encoding a ParB/Spo0J-like protein that is required for expression of genes in the sbn operon from sbnD onward. Expression of sbnD-I is drastically decreased in an sbnI mutant, and the mutant does not synthesize detectable SB during early phases of growth. Thus, SB-mediated iron acquisition is impaired in an sbnI mutant strain. We show that the protein forms dimers and tetramers in solution and binds to DNA within the sbnC coding region. Moreover, we show that SbnI binds heme and that heme-bound SbnI does not bind DNA. Finally, we show that providing exogenous heme to S. aureus growing in an iron-free medium results in delayed synthesis of SB. This is the first study in S. aureus that identifies a DNA-binding regulatory protein that senses heme to control gene expression for siderophore synthesis.

Protein design is a powerful tool for interrogating the basic requirements for the function of a metal site in a way that allows for the selective incorporation of elements that are important for function. Rubredoxins are small electron transfer proteins with a reduction potential centered near 0 mV (vs normal hydrogen electrode). All previous attempts to design a rubredoxin site have focused on incorporating the canonical CXXC motifs in addition to reproducing the peptide fold or using flexible loop regions to define the morphology of the site. We have produced a rubredoxin site in an utterly different fold, a three-helix bundle. The spectra of this construct mimic the ultraviolet–visible, Mössbauer, electron paramagnetic resonance, and magnetic circular dichroism spectra of native rubredoxin. Furthermore, the measured reduction potential suggests that this rubredoxin analogue could function similarly. Thus, we have shown that an α-helical scaffold sustains a rubredoxin site that can cycle with the desired potential between the Fe(II) and Fe(III) states and reproduces the spectroscopic characteristics of this electron transport protein without requiring the classic rubredoxin protein fold.

Copper proteins have the capacity to serve as both redox active catalysts and purely electron transfer centers. A longstanding question in this field is how the function of histidine ligated Cu centers are modulated by δ vs ε-nitrogen ligation of the imidazole. Evaluating the impact of these coordination modes on structure and function by comparative analysis of deposited crystal structures is confounded by factors such as differing protein folds and disparate secondary coordination spheres that make direct comparison of these isomers difficult. Here, we present a series of de novo designed proteins using the noncanonical amino acids 1-methyl-histidine and 3methyl-histidine to create Cu nitrite reductases where δor ε-nitrogen ligation is enforced by the opposite nitrogen's methylation as a means of directly comparing these two ligation states in the same protein fold. We find that ε-nitrogen ligation allows for a better nitrite reduction catalyst, displaying 2 orders of magnitude higher activity than the δ-nitrogen ligated construct. Methylation of the δ nitrogen, combined with a secondary sphere mutation we have previously published, has produced a new record for efficiency within a homogeneous aqueous system, improving by 1 order of magnitude the previously published most efficient construct. Furthermore, we have measured Michaelis-Menten kinetics on these highly active constructs, revealing that the remaining barriers to matching the catalytic efficiency (k cat /K M) of native Cu nitrite reductase involve both substrate binding (K M) and catalysis (k cat).

One of the hallmark advances in our understanding of metalloprotein function is showcased in our ability to design new, non-native, catalytically active protein scaffolds. This review highlights progress and milestone achievements in the field of de novo metalloprotein design focused on reports from the past decade with special emphasis on de novo designs couched within common subfields of bioinorganic study: heme binding proteins, monometal- and dimetal-containing catalytic sites, and metal-containing electron transfer sites. Within each subfield, we highlight several of what we have identified as significant and important contributions to either our understanding of that subfield or de novo metalloprotein design as a discipline. These reports are placed in context both historically and scientifically. General suggestions for future directions that we feel will be important to advance our understanding or accelerate discovery are discussed.

Keywords: enzymes ·h elical structures · metalloproteins · protein design · transition metals Angewandte Chemie Reviews 7678 www.angewandte.org