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Stabilizing bound O2 in myoglobin by valine68 (E11) to asparagine substitution.

Krzywda S, Murshudov GN, Brzozowski AM, Jaskolski M, Scott EE, Klizas SA, Gibson QH, Olson JS, Wilkinson AJ
Biochemistry 37 15896-907 (1998)
Related entries: 1m6m, 1mdn, 1mno, 1mwc, 1mwd

Cited: 16 times
EuropePMC logo PMID: 9843395

Abstract

The isopropyl side chain of valine68 in myoglobin has been replaced by the acetamide side chain of asparagine in an attempt to engineer higher oxygen affinity. The asparagine replacement introduces a second hydrogen bond donor group into the distal heme pocket which could further stabilize bound oxygen. The Val68 to Asn substitution leads to approximately 3-fold increases in oxygen affinity and 4-6-fold decreases in CO affinity. As a result, the M-value (KCO/KO2) is lowered 15-20-fold to a value close to unity. An even larger enhancement of O2 affinity is seen when asparagine68 is inserted into H64L sperm whale myoglobin which lacks a distal histidine. The overall rate constants for oxygen and carbon monoxide binding to the single V68N myoglobin mutants are uniformly lower than those for the wild-type protein. In contrast, the overall rate constant for NO association is unchanged. Analyses of time courses monitoring the geminate recombination of ligands following nanosecond and picosecond flash photolysis of MbNO and MbO2 indicate that the barrier to ligand binding from within the heme pocket has been raised with little effect on the barrier to diffusion of the ligand into the pocket from the solvent. The crystal structures of the aquomet, deoxy, oxy, and carbon monoxy forms of the V68N mutant have been determined to resolutions ranging from 1.75 to 2.2 A at 150 K. The overall structures are very similar to those of the wild-type protein with the principal alterations taking place within and around the distal heme pocket. In all four structures the asparagine68 side chain lies almost parallel to the plane of the heme with its amide group directed toward the back of the distal heme pocket. The coordinated water molecule in the aquomet form and the bound oxygen in the oxy form can form hydrogen-bonding interactions with both the Asn68 amide group and the imidazole side chain of His64. Surprisingly, in the carbon monoxy form of the V68N mutant, the histidine64 side chain has swung completely out the distal pocket, its place being taken by two ordered water molecules. Overall, these functional and structural results show that the asparagine68 side chain (i) forms a strong hydrogen bond with bound oxygen through its -NH2 group but (ii) sterically hinders the approach of ligands to the iron from within the distal heme pocket.

Articles - 1m6c mentioned but not cited (2)

  1. Effects of local protein environment on the binding of diatomic molecules to heme in myoglobins. DFT and dispersion-corrected DFT studies. Liao MS, Huang MJ, Watts JD. J Mol Model 19 3307-3323 (2013)
  2. Water stabilizes an alternate turn conformation in horse heart myoglobin. Bronstein A, Marx A. Sci Rep 13 6094 (2023)


Articles citing this publication (14)

  1. Hydration of protein-protein interfaces. Rodier F, Bahadur RP, Chakrabarti P, Janin J. Proteins 60 36-45 (2005)
  2. Application of the PM6 method to modeling proteins. Stewart JJ. J Mol Model 15 765-805 (2009)
  3. Distal heme pocket regulation of ligand binding and stability in soybean leghemoglobin. Kundu S, Hargrove MS. Proteins 50 239-248 (2003)
  4. Crystal structures of manganese- and cobalt-substituted myoglobin in complex with NO and nitrite reveal unusual ligand conformations. Zahran ZN, Chooback L, Copeland DM, West AH, Richter-Addo GB. J Inorg Biochem 102 216-233 (2008)
  5. Ligand migration in human myoglobin: steric effects of isoleucine 107(G8) on O(2) and CO binding. Ishikawa H, Uchida T, Takahashi S, Ishimori K, Morishima I. Biophys J 80 1507-1517 (2001)
  6. Using Biosynthetic Models of Heme-Copper Oxidase and Nitric Oxide Reductase in Myoglobin to Elucidate Structural Features Responsible for Enzymatic Activities. Bhagi-Damodaran A, Petrik I, Lu Y. Isr J Chem 56 773-790 (2016)
  7. Characterization and kinetics studies of water buffalo (Bubalus bubalis) myoglobin. Dosi R, Di Maro A, Chambery A, Colonna G, Costantini S, Geraci G, Parente A. Comp Biochem Physiol B Biochem Mol Biol 145 230-238 (2006)
  8. Human myoglobin recognition of oxygen: dynamics of the energy landscape. Wang Y, Baskin JS, Xia T, Zewail AH. Proc Natl Acad Sci U S A 101 18000-18005 (2004)
  9. Optical detection of disordered water within a protein cavity. Goldbeck RA, Pillsbury ML, Jensen RA, Mendoza JL, Nguyen RL, Olson JS, Soman J, Kliger DS, Esquerra RM. J Am Chem Soc 131 12265-12272 (2009)
  10. Myoglobinopathy is an adult-onset autosomal dominant myopathy with characteristic sarcoplasmic inclusions. Olivé M, Engvall M, Ravenscroft G, Cabrera-Serrano M, Jiao H, Bortolotti CA, Pignataro M, Lambrughi M, Jiang H, Forrest ARR, Benseny-Cases N, Hofbauer S, Obinger C, Battistuzzi G, Bellei M, Borsari M, Di Rocco G, Viola HM, Hool LC, Cladera J, Lagerstedt-Robinson K, Xiang F, Wredenberg A, Miralles F, Baiges JJ, Malfatti E, Romero NB, Streichenberger N, Vial C, Claeys KG, Straathof CSM, Goris A, Freyer C, Lammens M, Bassez G, Kere J, Clemente P, Sejersen T, Udd B, Vidal N, Ferrer I, Edström L, Wedell A, Laing NG. Nat Commun 10 1396 (2019)
  11. Dynamical comparison between myoglobin and hemoglobin. Aharoni R, Tobi D. Proteins 86 1176-1183 (2018)
  12. A heme pocket aromatic quadrupole modulates gas binding to cytochrome c'-β: Implications for NO sensors. Adams HR, Svistunenko DA, Wilson MT, Fujii S, Strange RW, Hardy ZA, Vazquez PA, Dabritz T, Streblow GJ, Andrew CR, Hough MA. J Biol Chem 299 104742 (2023)
  13. Data on the application of the molecular vector machine model: A database of protein pentafragments and computer software for predicting and designing secondary protein structures. Karasev V. Data Brief 28 104815 (2020)
  14. Burial of nonpolar surface area and thermodynamic stabilization of globins as a function of chain elongation. Jennaro TS, Beaty MR, Kurt-Yilmaz N, Luskin BL, Cavagnero S. Proteins 82 2318-2331 (2014)


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