The crystal structure of a ternary protein complex has been determined at 2.4 angstrom resolution. The complex is composed of three electron transfer proteins from Paracoccus denitrificans, the quinoprotein methylamine dehydrogenase, the blue copper protein amicyanin, and the cytochrome c551i. The central region of the c551i is folded similarly to several small bacterial c-type cytochromes; there is a 45-residue extension at the amino terminus and a 25-residue extension at the carboxyl terminus. The methylamine dehydrogenase-amicyanin interface is largely hydrophobic, whereas the amicyanin-cytochrome interface is more polar, with several charged groups present on each surface. Analysis of the simplest electron transfer pathways between the redox partners points out the importance of other factors such as energetics in determining the electron transfer rates.

The crystal structure of the complex between the quinoprotein methylamine dehydrogenase (MADH) and the type I blue copper protein amicyanin, both from Paracoccus denitrificans, has been determined at 2.5-A resolution using molecular replacement. The search model was MADH from Thiobacillus versutus. The amicyanin could be located in an averaged electron density difference map and the model improved by refinement and model building procedures. Nine beta-strands are observed within the amicyanin molecule. The copper atom is located between three antiparallel strands and is about 2.5 A below the protein surface. The major intermolecular interactions occur between amicyanin and the light subunit of MADH where the interface is largely hydrophobic. The copper atom of amicyanin and the redox cofactor of MADH are about 9.4 A apart. One of the copper ligands, His 95, lies between the two redox centers and may facilitate electron transfer between them.

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Methylamine dehydrogenase from Paracoccus denitrificans was purified to homogeneity in two steps from the periplasmic fraction of methylamine-grown cells. The enzyme exhibited a pI value of 4.3 and was composed of two 46,700-dalton subunits and two 15,500-dalton subunits. Each small subunit possessed a covalently bound pyrrolo-quinoline quinone prosthetic group. The amino acid compositions of the large and small subunits are very similar to those of other methylamine dehydrogenases which have been isolated from taxonomically different sources. It has been established recently that certain oxidoreductases from a variety of sources contain pyrrolo-quinoline quinone (PQQ) as a prosthetic group (2, 4, 10, 11). These quinoproteins include bacterial methanol (9) and glucose dehydrogenases (12), which possess noncovalently associated PQQ, and bacterial methylamine dehydrogenase, which contains a covalently bound form of PQQ (8,21). Mammalian plasma amine oxidase (26) and choline dehydrogenase (3) also possess covalently attached PQQ. When grown on methylamine as a sole source of carbon and energy, Paracoccus denitrificans synthesizes a methylamine dehydrogenase which functions in the periplasm of this gramnegative bacterium and donates electrons to a periplasmic type I blue copper protein, amicyanin (16). We have previously reported the partial purification of methylamine dehydrogenase from P. denitrificans (16) and have characterized the physical and redox properties of amicyanin (14,16,18,25) and periplasmic c-type cytochromes (14, 17) which accept electrons from methylamine dehydrogenase via amicyanin. This paper reports a relatively simple procedure by which methylamine dehydrogenase was purified to homogeneity from P. denitrificans and describes several of the physical and kinetic properties of this enzyme. MATERIALS AND METHODSBacterial strains and culture conditions. P. denitrificans (ATCC 13543) was grown aerobically at 30°C in the medium of Kornberg and Morris (23), supplemented with 0.05% NaHCO3, 0.01% yeast extract, and 0.5% methylamine, 0.5% methanol, or 0.3% succinate.Preparation of proteins. Methylamine dehydrogenase was purified from the periplasmic fraction of methylamine-grown cells, which was prepared by the method of Alefounder and Ferguson (1). Routinely, 15 to 20 g (wet weight) of cells was * Corresponding author. fractionated at a time, and four such preparations were pooled, concentrated by ultrafiltration over an Amicon YM5 membrane (Amicon Corp., Lexington, Mass.), dialyzed against 20 mM potassium phosphate (pH 7.2), and applied to a DEAE-cellulose column (3.5 by 30 cm) which had been equilibrated in the same buffer that was used for dialysis. This column was eluted with a linear gradient (2.0 liters) of 0 to 400 mM NaCl in the starting buffer. Fractions containing methylamine dehydrogenase were pooled, concentrated by ultrafiltration over an Amicon PM 30 membrane, dialyzed against 20 mM potassium phosphate (pH 7.2), and applied to a DEAE-Trisacryl (LKB Instruments, Inc., Rockville, Md.) column...