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

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 (T_m) 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.