Calprotectin (CP) is an antimicrobial protein produced and released by neutrophils that inhibits the growth of pathogenic microorganisms by sequestering essential metal nutrients in the extracellular space. In this work, spectroscopic and thermodynamic metal-binding studies are presented to delineate the zinc-binding properties of CP. Unique optical absorption and EPR spectroscopic signatures for the interfacial His3Asp and His4 sites of human calprotectin are identified by using Co(II) as a spectroscopic probe. Zinc competition titrations employing chromophoric Zn(II) indicators provide a 2:1 Zn(II):CP stoichiometry, confirm that the His3Asp and His4 sites of CP coordinate Zn(II), and reveal that the Zn(II) affinity of both sites is calcium-dependent. The calcium-insensitive Zn(II) competitor ZP4 afforded dissociation constants of Kd1 = 133 ± 58 pM and Kd2 = 185 ± 219 nM for CP in the absence of Ca(II). These values decreased to Kd1 ≤ 10 pM and Kd2 ≤ 240 pM in the presence of excess Ca(II). The Kd1 and Kd2 values are assigned to the His3Asp and His4 sites, respectively. In vitro antibacterial activity assays indicate that the metal-binding sites and Ca(II)-replete conditions are required for CP to inhibit the growth of both Gram-negative and –positive bacteria. Taken together, these data provide a working model whereby calprotectin responds to physiological Ca(II) gradients to become a potent Zn(II) chelator in the extracellular space.

Fe III -O 2 •− intermediates are well known in heme enzymes, but none have been characterized in the nonheme mononuclear Fe II enzyme family. Many steps in the O 2 activation and reaction cycle of Fe II -containing homoprotocatechuate 2,3-dioxygenase are made detectable by using the alternative substrate 4-nitrocatechol (4NC) and mutation of the active site His200 to Asn (H200N). Here, the first intermediate (Int-1) observed after adding O 2 to the H200N-4NC complex is trapped and characterized using EPR and Möss-bauer (MB) spectroscopies. Int-1 is a high-spin ( (1)(2)(3)(4)(5)(6)(7)(8). Internal electron transfer to form an Fe III -superoxo species converts the kinetically inert triplet ground state of O 2 to a doublet that can participate in the many types of chemistry characteristic of this mechanistically diverse group of enzymes. The same strategy is usually employed by heme-containing oxygenases and oxidases, leading in some cases to comparatively stable Fe III -superoxo intermediates that have been structurally and spectroscopically characterized (9-12). Instability of the putative superoxo intermediate in all mononuclear nonheme iron-containing enzymes has prevented similar characterization, although a superoxide level species has been reported for the dinuclear iron site of myo-inositol oxygenase (13).In recent studies of the nonheme Fe II -containing homoprotocatechuate 2,3-dioxygenase (2,3-HPCD), we have shown that three intermediates of the catalytic cycle can be trapped in one crystal for structural analysis (14). One of these intermediates has been proposed to be an Fe II -superoxo species based on the long Fe-O bond distances and an unexpected lack of planarity of the aromatic ring of the alternative substrate 4-nitrocatechol (4NC), which chelates the iron in ligand sites adjacent to that of the O 2 . In accord with the mechanism postulated for this enzyme class as illustrated in Scheme 1 (1, 8, 15-21), we have proposed that net electron transfer from 4NC through the Fe II to O 2 forms adjacent substrate and oxygen radicals (Scheme 1B). Recombination of the radicals would begin the ring cleavage and oxygen insertion reactions of this enzyme that eventually yield a muconic semialdehyde adduct as the product. A localized radical on the 4NC semiquinone at the incipient position of oxygen attack would account for the lack of ring planarity. Although this is the only structurally characterized nonheme Fe-superoxo species, the iron oxidation state differs from all of the other postulated Fe-superoxo intermediates.The mechanism that emerges from the structural and kinetic studies does not require a change in metal oxidation state to form a reactive intermediate (22). However, our studies of 2,3-HPCD in which Fe II is replaced with Mn II suggest that transient formaScheme 1. Proposed mechanism for extradiol dioxygenases. In the case of 2,3-HPCD, R is −CH 2 COO − and B is His200. When R is −NO 2 and His200 is changed to Asn, the reaction stalls before reaching intermediate C. Peroxide is slowly released a...

Calprotectin (CP) is a transition metal-chelating antimicrobial protein of the calcium-binding S100 family that is produced and released by neutrophils. It inhibits the growth of various pathogenic microorganisms by sequestering the transition metal ions manganese and zinc. In this work, we investigate the manganese-binding properties of calprotectin. We demonstrate that the unusual His4 motif (site 2) formed at the S100A8/S100A9 dimer interface is the site of high-affinity Mn(II) coordination. We identify a low-temperature Mn(II) spectroscopic signal for this site consistent with an octahedral Mn(II) coordination sphere with simulated zero-field splitting parameters D = 270 MHz and E/D = 0.33 (E = 81 MHz). This analysis, combined with studies of mutant proteins, suggests that four histidine residues (H17 and H27 of S100A8; H91 and H95 of S100A9) coordinate Mn(II) in addition to two as-yet unidentified ligands. The His3Asp motif (site 1), which is also formed at the S100A8/S100A9 dimer interface, does not provide a high-affinity Mn(II) binding site. Calcium binding to the EF-hand domains of CP increases the Mn(II) affinity of the His4 site from the low-micromolar to the mid-nanomolar range. Metal-ion selectivity studies demonstrate that CP prefers to coordinate Zn(II) over Mn(II). Nevertheless, the specificity of Mn(II) for the His4 site provides CP with the propensity to form mixed Zn:Mn:CP complexes where one Zn(II) ion occupies site 1 and one Mn(II) ion occupies site 2. These studies support the notion that CP responds to physiological calcium ion gradients to become a high-affinity transition metal ion chelator in the extracellular space where it inhibits microbial growth.

Substrates homoprotocatechuate (HPCA) and O2 bind to the FeII of Homoprotocatechuate 2,3-dioxygenase (FeHPCD) in adjacent coordination sites. Transfer of an electron(s) from HPCA to O2 via the iron is proposed to activate the substrates for reaction with each other to initiate aromatic ring cleavage. Here, rapid-freeze-quench methods are used to trap and spectroscopically characterize intermediates in the reactions of the HPCA complexes of FeHPCD and the variant His200Asn (FeHPCD-HPCA and H200N-HPCA) with O2. A blue intermediate forms within 20 ms after mixing O2 with H200N-HPCA (H200NInt1HPCA). Parallel mode EPR and Mössbauer spectroscopies show that this intermediate contains high-spin FeIII (S=5/2) antiferromagnetically coupled to a radical (SR=1/2) to yield an S=2 state. Together, optical and Mössbauer spectra of the intermediate support assignment of the radical as an HPCA semiquinone, implying that oxygen is bound as a (hydro)peroxo ligand. H200NInt1HPCA decays over the next 2 s, possibly through an FeII intermediate (H200NInt2HPCA), to yield product and the resting FeII enzyme. Reaction of FeHPCD-HPCA with O2 results in rapid formation of a colorless FeII intermediate (FeHPCDInt1HPCA). This species decays within 1 s to yield the product and the resting enzyme. The absence of a chromophore from a semiquinone or evidence for a spin-coupled species in FeHPCDInt1HPCA suggests it is an intermediate occurring after O2 activation and attack. The similar Mössbauer parameters for FeHPCDInt1HPCA and H200NInt2HPCA suggest these are similar intermediates. The results show that electron transfer from the substrate to the O2 via the iron does occur leading to aromatic ring cleavage.

Human defensin 5 (HD5) is a 32-residue cysteine-rich host-defense peptide that exhibits three disulfide bonds in the oxidized form (HD5ox). It is abundant in small intestinal Paneth cells, which release HD5 into the intestinal lumen and house a labile Zn(II) store of unknown function. Here we consider the redox properties of HD5 and report that the reduced form, HD5red, is a metal-ion chelator. HD5 has a midpoint potential of −257 mV at pH 7.0. HD5red utilizes its cysteine residues to coordinate one equivalent of Zn(II) with an apparent Kd1 value in the mid-picomolar range. Zn(II) or Cd(II) binding perturbs the oxidative folding pathway of HD5red to HD5ox. Whereas HD5red is highly susceptible to proteolytic degradation, the Zn(II)-bound form displays resistance to hydrolytic breakdown by trypsin and other proteases. The ability of a reduced defensin peptide to coordinate Zn(II) provides a putative mechanism for how these peptides persist in vivo.