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Moving Iron through ferritin protein nanocages depends on residues throughout each four ?-helix bundle subunit.

ABSTRACT: Eukaryotic H ferritins move iron through protein cages to form biologically required, iron mineral concentrates. The biominerals are synthesized during protein-based Fe²?/O? oxidoreduction and formation of [Fe³?O](n) multimers within the protein cage, en route to the cavity, at sites distributed over ~50 ?. Recent NMR and Co²?-protein x-ray diffraction (XRD) studies identified the entire iron path and new metal-protein interactions: (i) lines of metal ions in 8 Fe²? ion entry channels with three-way metal distribution points at channel exits and (ii) interior Fe³?O nucleation channels. To obtain functional information on the newly identified metal-protein interactions, we analyzed effects of amino acid substitution on formation of the earliest catalytic intermediate (diferric peroxo-A(650 nm)) and on mineral growth (Fe³?O-A(350 nm)), in A26S, V42G, D127A, E130A, and T149C. The results show that all of the residues influenced catalysis significantly (p < 0.01), with effects on four functions: (i) Fe²? access/selectivity to the active sites (Glu¹³?), (ii) distribution of Fe²? to each of the three active sites near each ion channel (Asp¹²?), (iii) product (diferric oxo) release into the Fe³?O nucleation channels (Ala²?), and (iv) [Fe³?O](n) transit through subunits (Val?², Thr¹??). Synthesis of ferritin biominerals depends on residues along the entire length of H subunits from Fe²? substrate entry at 3-fold cage axes at one subunit end through active sites and nucleation channels, at the other subunit end, inside the cage at 4-fold cage axes. Ferritin subunit-subunit geometry contributes to mineral order and explains the physiological impact of ferritin H and L subunits.

SUBMITTER: Haldar S

PROVIDER: S-EPMC3138320 | biostudies-literature | 2011 Jul

REPOSITORIES: biostudies-literature

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Moving Iron through ferritin protein nanocages depends on residues throughout each four α-helix bundle subunit.

Haldar Suranjana S Bevers Loes E LE Tosha Takehiko T Theil Elizabeth C EC

The Journal of biological chemistry 20110518 29

Eukaryotic H ferritins move iron through protein cages to form biologically required, iron mineral concentrates. The biominerals are synthesized during protein-based Fe²⁺/O₂ oxidoreduction and formation of [Fe³⁺O](n) multimers within the protein cage, en route to the cavity, at sites distributed over ~50 Å. Recent NMR and Co²⁺-protein x-ray diffraction (XRD) studies identified the entire iron path and new metal-protein interactions: (i) lines of metal ions in 8 Fe²⁺ ion entry channels with three ...[more]

PMID: 21592958

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Project description:De novo protein design represents an attractive approach for testing and extending our understanding of metalloprotein structure and function. Here, we describe our work on the design of DF (Due Ferri or two-iron in Italian), a minimalist model for the active sites of much larger and more complex natural diiron and dimanganese proteins. In nature, diiron and dimanganese proteins protypically bind their ions in 4-Glu, 2-His environments, and they catalyze diverse reactions, ranging from hydrolysis, to O2-dependent chemistry, to decarbonylation of aldehydes. In the design of DF, the position of each atom-including the backbone, the first-shell ligands, the second-shell hydrogen-bonded groups, and the well-packed hydrophobic core-was bespoke using precise mathematical equations and chemical principles. The first member of the DF family was designed to be of minimal size and complexity and yet to display the quintessential elements required for binding the dimetal cofactor. After thoroughly characterizing its structural, dynamic, spectroscopic, and functional properties, we added additional complexity in a rational stepwise manner to achieve increasingly sophisticated catalytic functions, ultimately demonstrating substrate-gated four-electron reduction of O2 to water. We also briefly describe the extension of these studies to the design of proteins that bind nonbiological metal cofactors (a synthetic porphyrin and a tetranuclear cluster), and a Zn2+/proton antiporting membrane protein. Together these studies demonstrate a successful and generally applicable strategy for de novo metalloprotein design, which might indeed mimic the process by which primordial metalloproteins evolved. We began the design process with a highly symmetrical backbone and binding site, by using point-group symmetry to assemble the secondary structures that position the amino acid side chains required for binding. The resulting models provided a rough starting point and initial parameters for the subsequent precise design of the final protein using modern methods of computational protein design. Unless the desired site is itself symmetrical, this process requires reduction of the symmetry or lifting it altogether. Nevertheless, the initial symmetrical structure can be helpful to restrain the search space during assembly of the backbone. Finally, the methods described here should be generally applicable to the design of highly stable and robust catalysts and sensors. There is considerable potential in combining the efficiency and knowledge base associated with homogeneous metal catalysis with the programmability, biocompatibility, and versatility of proteins. While the work reported here focuses on testing and learning the principles of natural metalloproteins by designing and studying proteins one at a time, there is also considerable potential for using designed proteins that incorporate both biological and nonbiological metal ion cofactors for the evolution of novel catalysts.

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Moving Iron through ferritin protein nanocages depends on residues throughout each four ?-helix bundle subunit.

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Moving Iron through ferritin protein nanocages depends on residues throughout each four α-helix bundle subunit.

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