David M. Mitchell

Rapid purification of wildtype and mutant cytochrome c oxidase from Rhodobacter sphaeroides by Ni2+-NTA affinity chromatography

A rapid and highly efficient method of purifying the aa 3‐type cytochrome c oxidase from Rhodobacter sphaeroides has been developed. This method relies upon a six‐histidine affinity tag fused to the C‐terminus of subunit I, which confers to the oxidase a high affinity for Ni2+‐nitrilotriacetic acid (NTA) agarose. The histidine‐tagged oxidase can be purified rapidly and with high yield by one affinity chromatography step, starting with solubilized membranes. The purified oxidase is >95% pure and possesses structural and functional characteristics of the wildtype enzyme. The six‐histidine tag can be easily added to pre‐constructed site‐directed mutants of subunit I, increasing the availability of purified cytochrome c oxidase mutants for biophysical and biochemical studies.

Matrix-assisted laser desorption ionization mass spectrometry of membrane proteins: Demonstration of a simple method to determine subunit molecular weights of hydrophobic subunits

Matrix-assisted laser desorption ionization (MALDI) mass spectrometry has been used to obtain accurate molecular weight information for each subunit of several hydrophobic integral membrane proteins: cytochrome bo3 (4 subunits) and cytochrome bd (2 subunits) from E. coli, and the bc1 complex (3 subunits) and the cytochrome c oxidase (3 subunits) from Rhodobacter sphaeroides. The results demonstrate that the MALDI method is a convenient, quick, sensitive and reliable means for obtaining the molecular masses of the subunits of purified multisubunit membrane proteins.

EPR studies of wild-type and several mutants of cytochrome c oxidase from Rhodobacter sphaeroides: Glu286 is not a bridging ligand in the cytochrome a3-CuB center

Wild‐type and several mutants of cytochrome c oxidase from Rhodobacter sphaeroides were characterized by EPR spectroscopy. A pH‐induced g12 signal, seen previously in mammalian cytochrome oxidase and assigned to the presence of a bridging car☐yl ligand in the bimetallic cytochrome a 3‐CuB site, is found also in the bacterial enzyme. Mutation of glutamate‐286 to glutamine inactivates the enzyme but does not affect this signal, demonstrating that the car☐yl group of this residue is not the bridging ligand. Three mutants, M106Q, located one helix turn below a histidine ligand to cytochrome a, and T352A as well as F391Q, located close to the bimetallic center, are shown to affect dramatically the low‐spin heme signal of cytochrome a. These mutants are essentially inactive, suggesting that these three mutations result in alterations to cytochrome a that render the oxidase non‐functional.

FT-IR analysis of membranes of Rhodobacter sphaeroides 2.4.3 grown under microaerobic and denitrifying conditions

Fourier transform infrared spectroscopic analysis of CO binding proteins in Rhodobacter sphaeroides reveals the presence of a membrane-bound nitric oxide reductase (Nor). Nor has been clearly distinguished from the cytochrome oxidases by the temperature-dependence of relaxation following photodissociation of the CO complex at cryogenic temperatures. The center frequency and band shape, 1970 cm-1 and 20-30 cm-1 width at half-peak height, are similar to those reported for resonance Raman spectra of purified Paracoccus denitrificans Nor. Additional evidence is presented to indicate this enzyme is part of dissimilatory nitric oxide metabolism and that one of the genes in the nor operon required for production of an active Nor is not required for protein assembly or heme incorporation.