Structure of an intermolecular electron-transfer complex: p-cresol methylhydroxylase at 6.0-A resolution

Area of Science:

• Biochemistry
• Structural Biology
• Enzymology

Background:

• p-Cresol methylhydroxylase (EC 1.17.99.1) is a flavocytochrome c involved in methyl-hydroxylating 4-cresol.
• Previous understanding suggested an alpha beta quaternary structure for this enzyme.

Purpose of the Study:

• To determine the high-resolution structure of p-cresol methylhydroxylase.
• To elucidate the enzyme's quaternary structure and subunit arrangement.

Main Methods:

• X-ray crystallography at 6.0-A resolution.
• Structure determination using two heavy-atom derivatives with anomalous scattering.
• Application of 2-fold averaging about a noncrystallographic axis.

Main Results:

• The enzyme exhibits an alpha 2 beta 2 tetrameric quaternary structure, differing from the previously proposed alpha beta form.
• The flavoprotein subunits (Mr ~49,000) form a tightly packed core, while smaller cytochrome subunits (Mr ~8,500) are located externally.
• The orientation and surface complementarity between cytochrome and flavoprotein subunits are clearly defined.

Conclusions:

• The determined alpha 2 beta 2 structure refutes the earlier alpha beta model.
• The arrangement of subunits provides a structural basis for understanding the enzyme's substrate titration and kinetic properties.
• The cytochrome subunit, while similar in size to other bacterial cytochromes, may represent a distinct subclass.