Design, synthesis, and characterization of a novel hemoprotein

Abstract

Here we describe a synthetic protein (6H7H) designed to bind four heme groups via bis-histidine axial ligation. The hemes are designed to bind perpendicular to another in an orientation that mimics the relative geometry of the two heme a groups in the active site of cytochrome c oxidase. Our newly developed protein-design program, called CORE, was implemented in the design of this novel hemoprotein. Heme titration studies resolved four distinct K D values (K D1 ‫ס‬ 80 nM, K D2 ‫ס‬ 18 nM, K D3 Ն 3 mM, K D4 Յ 570 nM, with K D3 × K D4 ‫ס‬ 1700); positive cooperativity in binding between the first and second heme, as well as substantial positive cooperativity between the third and forth heme, was observed. Chemical and thermal denaturation studies reveal a stable protein with native-like properties. Visible circular dichroism spectroscopy of holo-6H7H indicates excitonic coupling between heme groups. Further electrochemical and spectroscopic characterization of the holo-protein support a structure that is consistent with the predefined target structure.Keywords: de novo proteins; heme; hemoprotein; protein design Supplemental material: See www.proteinscience.org.Recent advances in theoretical, experimental, and computational protein design have led to the synthesis of metalloproteins with targeted function (Mutz et al. 1996(Mutz et al. , 1999Pinto et al., 1997;Benson et al. 1998;. A class of designed metalloproteins receiving considerable attention is hemoproteins (Sasaki and Kaiser 1989;Choma et al. 1994;Robertson et al. 1994;Rabanal et al. 1996;Gibney et al. 1997;Rojas et al. 1997aRojas et al. , 1997b. Native hemoproteins such as cytochromes, globins, and peroxidases perform a wide range of cellular function including electron transfer, oxygen transport, and catalysis. It is this diversity of reactivity that has been a driving force for the de novo design of functional hemoproteins. These designed proteins offer the potential for robust, highly tunable structures that are likely to provide ideal systems in which native hemoproteins can be better understood. An understanding of the role that ligands and local heme environment play in controlling the redox and catalytic reactivity of bound heme groups will ultimately lead to the construction of synthetic hemoproteins with novel targeted functionality.Combinatorial and so-called rational design methods have shown promise in producing synthetic proteins with predefined structures. Development of computational methods (Hellinga and Richards 1994; Desjarlais and Handel 1995a,b;Mayo 1996, 1997;Lazar et al. 1997;Su and Mayo 1997;Street and Mayo 1999;Jiang et al. 2000) has further expanded the ability to design synthetic proteins with targeted structure and function. However, despite the available tools, there still remains only a small number of uniquely folded designed hemoproteins. In many examples, the flexibility often associated with synthetic proteins precludes detailed characterization of structure-function relationships.We have implemented a computational...

Design and fine-tuning redox potentials of metalloproteins involved in electron transfer in bioenergetics

“…Interestingly, one of these models showed cooperativity between heme potentials [78,79], a well-known feature of multi-heme proteins. The CORE algorithm was developed to aid the design of native-like bundles that can bind to heme [87,88]. Another example of a de novo heme binding protein is the design of sequences that can fold into a globin fold and bind to heme in a 6-coordinated manner [89,90] (Figure 6b).…”

Mimicking Photosynthesis in a Computationally Designed Synthetic Metalloprotein

“…6 This value is more positive than most other synthetic hemoproteins with bis-histidine-bound heme groups. This suggests a more effective shielding of the heme group from water, consistent with the observed tight heme binding.…”

Structure and Reactivity of Hexacoordinate Hemoglobins

“…Other heme proteins with bis-histidyl coordination do not bind ligands [109,110], and a hexacoordinate Mb mutant protein is structurally constrained, and limited in its ability to bind exogenous ligands [53]. Thus, there has been interest in observing the structural changes accompanying ligand binding in the context of naturally occurring hxHbs.…”