Design, Synthesis, and Characterization of a Novel Hemoprotein

Overview

Journal Protein Sci

Specialty Biochemistry

Date 2001 Mar 27

PMID 11266610

Citations 5

Authors

Z Xu

R S Farid

Affiliations

Soon will be listed here.

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) = 80 nM, K(D2) = 18 nM, K(D3) > or = 3 mM, K(D4) < or = 570 nM, with K(D3) x K(D4) = 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.

Citing Articles

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

Hosseinzadeh P, Lu Y Biochim Biophys Acta. 2015; 1857(5):557-581.

PMID: 26301482 PMC: 4761536. DOI: 10.1016/j.bbabio.2015.08.006.

Metalloproteins containing cytochrome, iron-sulfur, or copper redox centers.

Liu J, Chakraborty S, Hosseinzadeh P, Yu Y, Tian S, Petrik I Chem Rev. 2014; 114(8):4366-469.

PMID: 24758379 PMC: 4002152. DOI: 10.1021/cr400479b.

Structure and reactivity of hexacoordinate hemoglobins.

Kakar S, Hoffman F, Storz J, Fabian M, Hargrove M Biophys Chem. 2010; 152(1-3):1-14.

PMID: 20933319 PMC: 4390088. DOI: 10.1016/j.bpc.2010.08.008.

Engineering heme binding sites in monomeric rop.

Di Nardo G, Di Venere A, Mei G, Sadeghi S, Wilson J, Gilardi G J Biol Inorg Chem. 2009; 14(4):497-505.

PMID: 19152012 DOI: 10.1007/s00775-009-0465-0.

Geometric constraints for porphyrin binding in helical protein binding sites.

Negron C, Fufezan C, Koder R Proteins. 2008; 74(2):400-16.

PMID: 18636480 PMC: 2844854. DOI: 10.1002/prot.22143.

References

Chen H, Sanyal G . Direct biophysical characterization of human apolipoprotein A-1 in ISCOMs. J Pharm Sci. 1999; 88(11):1122-6. DOI: 10.1021/js990158l. View

Street A, Mayo S . Computational protein design. Structure. 1999; 7(5):R105-9. DOI: 10.1016/s0969-2126(99)80062-8. View

Hsu M, Woody R . The origin of the heme Cotton effects in myoglobin and hemoglobin. J Am Chem Soc. 1971; 93(14):3515-25. DOI: 10.1021/ja00743a036. View

Burstein E, Vedenkina N, Ivkova M . Fluorescence and the location of tryptophan residues in protein molecules. Photochem Photobiol. 1973; 18(4):263-79. DOI: 10.1111/j.1751-1097.1973.tb06422.x. View

Stellwagen E . Haem exposure as the determinate of oxidation-reduction potential of haem proteins. Nature. 1978; 275(5675):73-4. DOI: 10.1038/275073a0. View

Chetverin A, Agalarov S, Emelyanenko V, Burstein E . Small differences in tryptophan fluorescence spectra of 'sodium' and 'potassium' forms of (Na+, K+)-dependent adenosinetriphosphatase. Eur J Biochem. 1980; 108(1):157-61. DOI: 10.1111/j.1432-1033.1980.tb04707.x. View

Kabsch W, Sander C . Dictionary of protein secondary structure: pattern recognition of hydrogen-bonded and geometrical features. Biopolymers. 1983; 22(12):2577-637. DOI: 10.1002/bip.360221211. View

WOOD P . Bacterial proteins with CO-binding b- or c-type haem. Functions and absorption spectroscopy. Biochim Biophys Acta. 1984; 768(3-4):293-317. DOI: 10.1016/0304-4173(84)90020-x. View

Degrado W, Wasserman Z, Lear J . Protein design, a minimalist approach. Science. 1989; 243(4891):622-8. DOI: 10.1126/science.2464850. View

10.

Lee F, Auld D, Vallee B . Tryptophan fluorescence as a probe of placental ribonuclease inhibitor binding to angiogenin. Biochemistry. 1989; 28(1):219-24. DOI: 10.1021/bi00427a030. View

11.

Kuwajima K . The molten globule state as a clue for understanding the folding and cooperativity of globular-protein structure. Proteins. 1989; 6(2):87-103. DOI: 10.1002/prot.340060202. View

12.

Semisotnov G, RODIONOVA N, Razgulyaev O, Uversky V, Gripas A, Gilmanshin R . Study of the "molten globule" intermediate state in protein folding by a hydrophobic fluorescent probe. Biopolymers. 1991; 31(1):119-28. DOI: 10.1002/bip.360310111. View

13.

Viguera A, Goni F, Rial E . The uncoupling protein from brown adipose tissue mitochondria. The environment of the tryptophan residues as revealed by quenching of the intrinsic fluorescence. Eur J Biochem. 1992; 210(3):893-9. DOI: 10.1111/j.1432-1033.1992.tb17493.x. View

14.

Hill B . The sequence of electron carriers in the reaction of cytochrome c oxidase with oxygen. J Bioenerg Biomembr. 1993; 25(2):115-20. DOI: 10.1007/BF00762853. View

15.

Babcock G, Varotsis C . Discrete steps in dioxygen activation--the cytochrome oxidase/O2 reaction. J Bioenerg Biomembr. 1993; 25(2):71-80. DOI: 10.1007/BF00762849. View

16.

BLAUER G, Sreerama N, Woody R . Optical activity of hemoproteins in the Soret region. Circular dichroism of the heme undecapeptide of cytochrome c in aqueous solution. Biochemistry. 1993; 32(26):6674-9. DOI: 10.1021/bi00077a021. View

17.

Genov N, Nicolov P, Betzel C, Wilson K, Dolashka P . Fluorescence properties of subtilisins and related proteinases (subtilases): relation to X-ray models. J Photochem Photobiol B. 1993; 18(2-3):265-72. DOI: 10.1016/1011-1344(93)80074-j. View

18.

Gilardi G, Mei G, Rosato N, Canters G, Finazzi-Agro A . Unique environment of Trp48 in Pseudomonas aeruginosa azurin as probed by site-directed mutagenesis and dynamic fluorescence spectroscopy. Biochemistry. 1994; 33(6):1425-32. DOI: 10.1021/bi00172a020. View

19.

Myszka D, Chaiken I . Design and characterization of an intramolecular antiparallel coiled coil peptide. Biochemistry. 1994; 33(9):2363-72. DOI: 10.1021/bi00175a003. View

20.

Robertson D, Farid R, Moser C, Urbauer J, Mulholland S, Pidikiti R . Design and synthesis of multi-haem proteins. Nature. 1994; 368(6470):425-32. DOI: 10.1038/368425a0. View