Resonance Raman Investigations of Site-Directed Mutants of Myoglobin: Effects of Distal Histidine Replacement

Dimitrios Morikis, Paul M. Champion, Barry A. Springer, Stephen G. Sligar

Research output: Contribution to journalArticlepeer-review

Abstract

The resonance Raman spectra of met-, deoxy-, and (carbonmonoxy)myoglobin (MbCO) are studied as a function of amino acid replacement at the distal histidine-E7 position. The synthetic wild type is found to be spectroscopically identical with the native material. The methionine and glycine replacements do not affect the met or deoxy spectra but do lead to distinct changes in the νFe-Co region of the MbCO spectrum. The native MbCO displays a pH-dependent population redistribution of the νFe-Co modes, while the analogous population in the mutant systems is found to be pH independent. This indicates that histidine-E7 is the titratable group in native MbCO. Moreover, the pH dependence of the population dynamics is found to be inconsistent with a simple two-state Henderson-Hasselbalch analysis. Instead, we suggest a four-state model involving the coupling of histidine protonation and conformational change. Within this model, the pK of the distal histidine is found to be 6.0 in the “open” configuration and 3.8 in the “closed” conformation. This corresponds to a 3 kcal/mol destabilization of the positively charged distal histidine within the hydrophobic pocket and suggests how protonation can lead to a larger population of the “open” conformation. At pH 7, the pocket is found to be “open” approximately 3% of the time. Further work, involving both IR and Raman measurements, allows the electron-nuclear coupling strengths of the various νFe-Co and νC-O Raman modes to be determined. The slowly rebinding conformational state, corresponding to νFe-Co = 518 cm-1C-O = 1932 cm-1), displays unusually weak coupling of the Fe-CO mode to the Soret transition. Studies of the νFe-Co region as a function of temperature reveal that the equilibria between the conformational states are quenched in both the native and glycine mutant below the freezing point of the solvent. Unusual line narrowing of the νFe-Co modes at the phase transition is also observed in all samples studied. This line narrowing stands in marked contrast to the other heme Raman modes and suggests that Fe-CO librational motion and/or distal pocket vibrational (or conformational) excitations are involved in the line broadening at room temperature.

Original languageEnglish (US)
Pages (from-to)4791-4800
Number of pages10
JournalBiochemistry
Volume28
Issue number11
DOIs
StatePublished - 1989

ASJC Scopus subject areas

  • Biochemistry

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