Effect of Heme Binding on the Structure and Stability of Escherichia coli Apocytochrome b562

Yiqing Feng, Stephen G. Sligar

Research output: Contribution to journalArticlepeer-review


The structure and stability of apocytochrome b562 were explored using absorption and circular dichroism spectroscopic methods. The polypeptide chain retains a well-defined structure when the prosthetic heme group is removed from cytochrome b562. Circular dichroism measurements estimate 60% helicity for apocytochrome b562, compared with 80% helicity found in holocytochrome b562. At low pH, apocytochrome b562 displays a midpoint pH of 2.9, while ferricytochrome b562 displays a midpoint pH of 2.3. The unfolding of the apoprotein by urea and heat can be well approximated by the two-state transition model. The stability of apocytochrome b562 is significantly reduced from that of the holoprotein. The free energy of stabilization (ΔG°) and the midpoint transition temperature (Tm) for apocytochrome b562 are found to be 3.2 ± 0.5 kcal/mol and 52.3 ± 0.9 °C, respectively, compared with 6.6 ± 0.5 kcal/mol and 67.2 ± 0.5 °C for ferricytochrome b562. The smaller heat capacity change upon unfolding of apocytochrome b562 than that of ferricytochrome b562, estimated from the thermodynamic parameters, indicates that apocytochrome b562 possesses a smaller hydrophobic core than holocytochrome b562. Size-exclusion chromatography studies indicate that the apoprotein is slightly more extended in molecular dimension than ferricytochrome b562. The data suggest that apocytochrome b562 resembles a “molten globule” or a “collapsed form” of the holoprotein, in which secondary structure formation is largely complete while the global folding is either only partially complete or dynamically expanded.

Original languageEnglish (US)
Pages (from-to)10150-10155
Number of pages6
Issue number42
StatePublished - Oct 1 1991

ASJC Scopus subject areas

  • Biochemistry


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