The CO stretching mode infrared spectrum of substrate-free cytochrome P-450cam-CO: The effect of solvent conditions, temperature, and pressure

Christiane Jung, Otto Ristau, Heike Schulze, Stephen G. Sligar

Research output: Contribution to journalArticlepeer-review

Abstract

The effect of pH, glycerol, temperature, and pressure on the carbon monoxide (CO) stretch mode of substrate-free cytochrome P-450cam (CYP101) was studied. Complex spectra of overlapping bands have been observed. CO stretch bands centered at about 1911-1918 cm1 (band I), 1927-1931 cm1 (band II), 1940-1942 cm1 (band III), 1950-1953 cm1 (band IV), 1960-1963 cm1 (band V) and 1966-1973 cm1 (band VI) are obtained from the fitting analyses independently of the lineshape model used. Only two or three bands are dominant in each spectrum. Compared to bands I, II and III, the bands IV and V are assigned to correspond to a weaker polar contact between the CO ligand and a polar group in the heme pocket (probably Thr252) because of the opposite effect of glycerol (osmotic pressure) and hydrostatic pressure on the intensity and frequency of these bands. The different CO stretch bands are interpreted as indicating conformational substates of the protein. It is suggested that water in the heme pocket plays an important role for the substate equilibrium. This substate equilibrium freezes in at the glass transition temperature, Tg, of the protein/solvent mixture. For the temperature region above Tg the thermodynamic parameters and volumes for the substates have been determined by a global fit analysis of the temperature- and pressure-dependent populations and they are compared to the respective values for camphor-bound cytochrome P-450cam.

Original languageEnglish (US)
Pages (from-to)660-669
Number of pages10
JournalEuropean Journal of Biochemistry
Volume235
Issue number3
DOIs
StatePublished - 1996

Keywords

  • Conformational substates
  • Cytochrome P-450
  • Infrared spectroscopy

ASJC Scopus subject areas

  • Biochemistry

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