For present purposes, a protein-bound metal site consists of one or more metal ions and all protein side chain and exogenous bridging and terminal ligands that define the first coordination sphere of each metal ion. Such sites can be classified into five basic types with the indicated functions: (1) structural -- configuration (in part) of protein tertiary and/or quaternary structure; (2) storage -- uptake, binding, and release of metals in soluble form: (3) electron transfer -- uptake, release, and storage of electrons; (4) dioxygen binding -- metal-O{sub 2} coordination and decoordination; and (5) catalytic -- substrate binding, activation, and turnover. The authors present here a classification and structure/function analysis of native metal sites based on these functions, where 5 is an extensive class subdivided by the type of reaction catalyzed. Within this purview, coverage of the various site types is extensive, but not exhaustive. The purpose of this exposition is to present examples of all types of sites and to relate, insofar as is currently feasible, the structure and function of selected types. The authors largely confine their considerations to the sites themselves, with due recognition that these site features are coupled to protein structure at all levels. In themore » next section, the coordination chemistry of metalloprotein sites and the unique properties of a protein as a ligand are briefly summarized. Structure/function relationships are systematically explored and tabulations of structurally defined sites presented. Finally, future directions in bioinorganic research in the context of metal site chemistry are considered. 620 refs.« less

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Structural and Functional Aspects of Metal Sites in Biology

Kidney bean purple acid phosphatase (KBPAP) is an Fe(III)-Zn(II) metalloenzyme resembling the mammalian Fe(III)-Fe(II) purple acid phosphatases. The structure of the homodimeric 111-kilodalton KBPAP was determined at a resolution of 2.9 angstroms. The enzyme contains two domains in each subunit. The active site is located in the carboxyl-terminal domain at the carboxy end of two sandwiched beta alpha beta alpha beta motifs. The two metal ions are 3.1 angstroms apart and bridged monodentately by Asp164. The iron is further coordinated by Tyr167, His325, and Asp135, and the zinc by His286, His323, and Asn201. The active-site structure is consistent with previous proposals regarding the mechanism of phosphate ester hydrolysis involving nucleophilic attack on the phosphate group by an Fe(III)-coordinated hydroxide ion.

http://science.sciencemag.org/content/sci/268/5216/1489.full.pdf

Crystal structure of a purple acid phosphatase containing a dinuclear Fe(III)-Zn(II) active site.

With the aim of critically assessing the current models for metal ion assisted hydrolytic reaction mechanisms, an updated review of the current understanding of metallohydrolase-catalyzed reactions is presented. Focus is on four systems, purple acid phosphatases (PAPs), Ser/Thr protein phosphatases (PPs), 3′-5′ exonucleases, and 5′-nucleotidases (5′-NTs), which have contributed to major advancement of the understanding of the catalytic mechanisms that operate in such enzymes.

/pdf/the-catalytic-mechanisms-of-binuclear-metallohydrolases-2gxxvk9jno.pdf

The catalytic mechanisms of binuclear metallohydrolases.

Mechanism of Fe III –Zn II purple acid phosphatase based on crystal structures

Di-iron-carboxylate proteins.

In order to perform Mossbauer studies, Zn(II) in the Fe(III)-Zn(II) purple acid phosphatase of the red kidney bean has been exchanged by incubating the semiapoenzyme with 57Fe(II). The resulting Fe(III)-57Fe(II) enzyme has 125% activity, compared with that of the Zn(II) enzyme. It can be oxidized by H2O2 or peroxydisulfate to the Fe(III)-57Fe(III) species with a 30-times lower activity. Incubation of the metal-free apoenzyme with 57Fe(II) in the presence of O2 leads to the 57Fe(III)-57Fe(II) species which is stable in dilute solutions, but partially oxidized during the concentration procedure to the 57Fe(III)-57Fe(III) enzyme. Limited reduction of the oxidized enzyme with ascorbate delivers a mixture of the Fe(II)-Fe(II)/Fe(III)-Fe(III) species, but not the mixed valent Fe(III)-Fe(II) species, indicating that after the transfer of the first electron the second electron of the ascorbate radical is immediately transferred to the second Fe(III).



The Mossbauer spectra of the oxidized species show at 4.2 K two quadrupole doublets with δ of 0.51 mm/s and 0.53 mm/s and E of 1.46 and 0.96 mm/s indicating high spin Fe(III) in two different binding sites, obviously with a higher asymmetry in the chromophoric Fe(III) site. The values are too low for a μ-oxo bridge. The mixed-valent Fe(III)-Fe(II) species shows two quadrupole doublet with δ values of 0.55 mm/s and 1.14 mm/s and E values of 1.43 mm/s and 3.01 mm/s at 70 K for high spin Fe(II) and Fe(III), but the signal of the Fe(II) component shows magnetic patterns at 4.2 K indicating a half-integer spin system with antiferromagnetic coupling. The Fe(II)-Fe(II) system exhibits two quadrupole doublets with δ values of 1.18 mm/s and 1.22 mm/s and with E values of 3.69 mm/s and 2.68 mm/s again indicating a higher asymmetry in the originally chromophoric Fe(III)-binding site. Addition of phosphate shows only minor differences in the oxidized enzyme and in the mixed valent Fe(III)-Fe(II) system. Interaction with O2 is discussed.

https://febs.onlinelibrary.wiley.com/doi/pdf/10.1111/j.1432-1033.1993.tb17926.x

Michael Körner

Papers

Zn‐exchange and Mössbauer studies on the [Fe‐Fe] derivatives of the purple acid Fe(III)‐Zn(II)‐phosphatase from kidney beans

Crystallization and preliminary crystallographic data of purple acid phosphatase from red kidney bean.