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Title: Introducing a 2-His-1-Glu Nonheme Iron Center into Myoglobin Confers Nitric Oxide Reductase Activity

Abstract

A conserved 2-His-1-Glu metal center, as found in natural nonheme iron-containing enzymes, was engineered into sperm whale myoglobin by replacing Leu29 and Phe43 with Glu and His, respectively (swMb L29E, F43H, H64, called Fe{sub B}Mb(-His)). A high resolution (1.65 {angstrom}) crystal structure of Cu(II)-CN?-Fe{sub B}Mb(-His) was determined, demonstrating that the unique 2-His-1-Glu metal center was successfully created within swMb. The Fe{sub B}Mb(-His) can bind Cu, Fe, or Zn ions, with both Cu(I)-Fe{sub B}Mb(-His) and Fe(II)-Fe{sub B}Mb(-His) exhibiting nitric oxide reductase (NOR) activities. Cu dependent NOR activity was significantly higher than that of Fe in the same metal binding site. EPR studies showed that the reduction of NO to N{sub 2}O catalyzed by these two enzymes resulted in different intermediates; a five-coordinate heme-NO species was observed for Cu(I)-Fe{sub B}Mb(-His) due to the cleavage of the proximal heme Fe-His bond, while Fe(II)-Fe{sub B}Mb(-His) remained six-coordinate. Therefore, both the metal ligand, Glu29, and the metal itself, Cu or Fe, play crucial roles in NOR activity. This study presents a novel protein model of NOR and provides insights into a newly discovered member of the NOR family, gNOR.

Authors:
; ; ; ; ; ;
Publication Date:
Research Org.:
Brookhaven National Lab. (BNL), Upton, NY (United States)
Sponsoring Org.:
DOE - OFFICE OF SCIENCE
OSTI Identifier:
1014316
Report Number(s):
BNL-93982-2010-JA
Journal ID: ISSN 0002-7863; R&D Project: BO-070; KP1605010; TRN: US201111%%269
DOE Contract Number:  
DE-AC02-98CH10886
Resource Type:
Journal Article
Journal Name:
Journal of American Chemical Society
Additional Journal Information:
Journal Volume: 132; Journal Issue: 29; Journal ID: ISSN 0002-7863
Country of Publication:
United States
Language:
English
Subject:
59 BASIC BIOLOGICAL SCIENCES; 99 GENERAL AND MISCELLANEOUS//MATHEMATICS, COMPUTING, AND INFORMATION SCIENCE; CETACEANS; CLEAVAGE; CRYSTAL STRUCTURE; ENZYMES; HEME; IRON; MYOGLOBIN; NITRIC OXIDE; OXIDOREDUCTASES; PROTEINS; RESOLUTION; SPERMATOZOA

Citation Formats

Lin, Y W, Robinson, H, Yeung, N, Gao, Y -G, Miner, K D, Lei, L, and Lu, Y. Introducing a 2-His-1-Glu Nonheme Iron Center into Myoglobin Confers Nitric Oxide Reductase Activity. United States: N. p., 2010. Web. doi:10.1021/ja103516n.
Lin, Y W, Robinson, H, Yeung, N, Gao, Y -G, Miner, K D, Lei, L, & Lu, Y. Introducing a 2-His-1-Glu Nonheme Iron Center into Myoglobin Confers Nitric Oxide Reductase Activity. United States. https://doi.org/10.1021/ja103516n
Lin, Y W, Robinson, H, Yeung, N, Gao, Y -G, Miner, K D, Lei, L, and Lu, Y. 2010. "Introducing a 2-His-1-Glu Nonheme Iron Center into Myoglobin Confers Nitric Oxide Reductase Activity". United States. https://doi.org/10.1021/ja103516n.
@article{osti_1014316,
title = {Introducing a 2-His-1-Glu Nonheme Iron Center into Myoglobin Confers Nitric Oxide Reductase Activity},
author = {Lin, Y W and Robinson, H and Yeung, N and Gao, Y -G and Miner, K D and Lei, L and Lu, Y},
abstractNote = {A conserved 2-His-1-Glu metal center, as found in natural nonheme iron-containing enzymes, was engineered into sperm whale myoglobin by replacing Leu29 and Phe43 with Glu and His, respectively (swMb L29E, F43H, H64, called Fe{sub B}Mb(-His)). A high resolution (1.65 {angstrom}) crystal structure of Cu(II)-CN?-Fe{sub B}Mb(-His) was determined, demonstrating that the unique 2-His-1-Glu metal center was successfully created within swMb. The Fe{sub B}Mb(-His) can bind Cu, Fe, or Zn ions, with both Cu(I)-Fe{sub B}Mb(-His) and Fe(II)-Fe{sub B}Mb(-His) exhibiting nitric oxide reductase (NOR) activities. Cu dependent NOR activity was significantly higher than that of Fe in the same metal binding site. EPR studies showed that the reduction of NO to N{sub 2}O catalyzed by these two enzymes resulted in different intermediates; a five-coordinate heme-NO species was observed for Cu(I)-Fe{sub B}Mb(-His) due to the cleavage of the proximal heme Fe-His bond, while Fe(II)-Fe{sub B}Mb(-His) remained six-coordinate. Therefore, both the metal ligand, Glu29, and the metal itself, Cu or Fe, play crucial roles in NOR activity. This study presents a novel protein model of NOR and provides insights into a newly discovered member of the NOR family, gNOR.},
doi = {10.1021/ja103516n},
url = {https://www.osti.gov/biblio/1014316}, journal = {Journal of American Chemical Society},
issn = {0002-7863},
number = 29,
volume = 132,
place = {United States},
year = {Wed Jul 28 00:00:00 EDT 2010},
month = {Wed Jul 28 00:00:00 EDT 2010}
}