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 (delta G degrees) and the midpoint transition temperature (Tm) for apocytochrome b562 are found to be 3.2 +/- 0.5 kcal/mol and 52.3 +/- 0.9 degrees C, respectively, compared with 6.6 +/- 0.5 kcal/mol and 67.2 +/- 0.5 degrees 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.
Feng
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YQ
Department of Biochemistry, University of Illinois, Urbana 61801.
Sligar
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D001426:Bacterial Proteins / Q000737:chemistry*; D002850:Chromatography, Gel; D002942:Circular Dichroism; D003573:Cytochrome b Group / Q000737:chemistry*; D004795:Enzyme Stability; D004926:Escherichia coli / Q000201:enzymology*; D029968:Escherichia coli Proteins; D006418:Heme / Q000737:chemistry* / Q000502:physiology; D006358:Hot Temperature; D006863:Hydrogen-Ion Concentration; D011485:Protein Binding; D011487:Protein Conformation
D001426:Bacterial Proteins; D003573:Cytochrome b Group; D029968:Escherichia coli Proteins; D006418:Heme; C017850:cytochrome b562, E coli