Structure at 2.7 Å resolution of the Paracoccus denitrificans two-subunit cytochrome c oxidase complexed with an antibody FV fragment

The aa3 type cytochrome c oxidase consisting of the core subunits I and II only was isolated from the soil bacterium Paracoccus denitrificans and crystallized as complex with a monoclonal antibody Fv fragment. Crystals could be grown in the presence of a number of different nonionic detergents. However, only undecyl-β-d-maltoside and cyclohexyl-hexyl-β-d-maltoside yielded well-ordered crystals suitable for high resolution x-ray crystallographic studies. The crystals belong to space group P212121 and diffract x-rays to at least 2.5 Å (1 Å = 0.1 nm) resolution using synchrotron radiation. The structure was determined to a resolution of 2.7 Å using molecular replacement and refined to a crystallographic R-factor of 20.5% (Rfree = 25.9%). The refined model includes subunits I and II and the 2 chains of the Fv fragment, 2 heme A molecules, 3 copper atoms, and 1 Mg/Mn atom, a new metal (Ca) binding site, 52 tentatively identified water molecules, and 9 detergent molecules. Only four of the water molecules are located in the cytoplasmic half of cytochrome c oxidase. Most of them are near the interface of subunits I and II. Several waters form a hydrogen-bonded cluster, including the heme propionates and the Mg/Mn binding site. The Fv fragment binds to the periplasmic polar domain of subunit II and is critically involved in the formation of the crystal lattice. The crystallization procedure is well reproducible and will allow for the analysis of the structures of mechanistically interesting mutant cytochrome c oxidases.

NADH-quinone oxidoreductase: PSST subunit couples electron transfer from iron–sulfur cluster N2 to quinone

The proton-translocating NADH-quinone oxidoreductase (EC 1.6.99.3) is the largest and least understood enzyme complex of the respiratory chain. The mammalian mitochondrial enzyme (also called complex I) contains more than 40 subunits, whereas its structurally simpler bacterial counterpart (NDH-1) in Paracoccus denitrificans and Thermus thermophilus HB-8 consists of 14 subunits. A major unsolved question is the location and mechanism of the terminal electron transfer step from iron–sulfur cluster N2 to quinone. Potent inhibitors acting at this key region are candidate photoaffinity probes to dissect NADH-quinone oxidoreductases. Complex I and NDH-1 are very sensitive to inhibition by a variety of structurally diverse toxicants, including rotenone, piericidin A, bullatacin, and pyridaben. We designed (trifluoromethyl)diazirinyl[3H]pyridaben ([3H]TDP) as our photoaffinity ligand because it combines outstanding inhibitor potency, a suitable photoreactive group, and tritium at high specific activity. Photoaffinity labeling of mitochondrial electron transport particles was specific and saturable. Isolation, protein sequencing, and immunoprecipitation identified the high-affinity specifically labeled 23-kDa subunit as PSST of complex I. Immunoprecipitation of labeled membranes of P. denitrificans and T. thermophilus established photoaffinity labeling of the equivalent bacterial NQO6. Competitive binding and enzyme inhibition studies showed that photoaffinity labeling of the specific high-affinity binding site of PSST is exceptionally sensitive to each of the high-potency inhibitors mentioned above. These findings establish that the homologous PSST of mitochondria and NQO6 of bacteria have a conserved inhibitor-binding site and that this subunit plays a key role in electron transfer by functionally coupling iron–sulfur cluster N2 to quinone.

The roles of the two proton input channels in cytochrome c oxidase from Rhodobacter sphaeroides probed by the effects of site-directed mutations on time-resolved electrogenic intraprotein proton transfer

The crystal structures of cytochrome c oxidase from both bovine and Paracoccus denitrificans reveal two putative proton input channels that connect the heme-copper center, where dioxygen is reduced, to the internal aqueous phase. In this work we have examined the role of these two channels, looking at the effects of site-directed mutations of residues observed in each of the channels of the cytochrome c oxidase from Rhodobacter sphaeroides. A photoelectric technique was used to monitor the time-resolved electrogenic proton transfer steps associated with the photo-induced reduction of the ferryl-oxo form of heme a3 (Fe4+ = O2−) to the oxidized form (Fe3+OH−). This redox step requires the delivery of a “chemical” H+ to protonate the reduced oxygen atom and is also coupled to proton pumping. It is found that mutations in the K channel (K362M and T359A) have virtually no effect on the ferryl-oxo-to-oxidized (F-to-Ox) transition, although steady-state turnover is severely limited. In contrast, electrogenic proton transfer at this step is strongly suppressed by mutations in the D channel. The results strongly suggest that the functional roles of the two channels are not the separate delivery of chemical or pumped protons, as proposed recently [Iwata, S., Ostermeier, C., Ludwig, B. & Michel, H. (1995) Nature (London) 376, 660–669]. The D channel is likely to be involved in the uptake of both “chemical” and “pumped” protons in the F-to-Ox transition, whereas the K channel is probably idle at this partial reaction and is likely to be used for loading the enzyme with protons at some earlier steps of the catalytic cycle. This conclusion agrees with different redox states of heme a3 in the K362M and E286Q mutants under aerobic steady-state turnover conditions.

Construction and characterization of an azurin analog for the purple copper site in cytochrome c oxidase.

A protein analog of a purple copper center has been constructed from a recombinant blue copper protein (Pseudomonas aeruginosa azurin) by replacing the loop containing the three ligands to the blue copper center with the corresponding loop of the CuA center in cytochrome c oxidase (COX) from Paracoccus denitrificans. The electronic absorption in the UV and visible region (UV-vis) and electron paramagnetic resonance (EPR) spectra of this analog are remarkably similar to those of the native CuA center in COX from Paracoccus denitrificans. The above spectra can be obtained upon addition of a mixture of Cu2+ and Cu+. Addition of Cu2+ only results in a UV-vis spectrum consisting of absorptions from both a purple copper center and a blue copper center. This spectrum can be converted to the spectrum of a pure purple copper by a prolonged incubation in the air, or by addition of excess ascorbate. The azurin mutant reported here is an example of an engineered purple copper center with the A480/A530 ratio greater than 1 and with no detectable hyperfines, similar to those of the CuA sites in COX of bovine heart and of Paracoccus denitrificans.

Isolation of a cDNA encoding human holocarboxylase synthetase by functional complementation of a biotin auxotroph of Escherichia coli.

Holocarboxylase synthetase (HCS) catalyzes the biotinylation of the four biotin-dependent carboxylases in human cells. Patients with HCS deficiency lack activity of all four carboxylases, indicating that a single HCS is targeted to the mitochondria and cytoplasm. We isolated 21 human HCS cDNA clones, in four size classes of 2.0-4.0 kb, by complementation of an Escherichia coli birA mutant defective in biotin ligase. Expression of the cDNA clones promoted biotinylation of the bacterial biotinyl carboxyl carrier protein as well as a carboxyl-terminal fragment of the alpha subunit of human propionyl-CoA carboxylase expressed from a plasmid. The open reading frame encodes a predicted protein of 726 aa and M(r) 80,759. Northern blot analysis revealed the presence of a 5.8-kb major species and 4.0-, 4.5-, and 8.5-kb minor species of poly(A)+ RNA in human tissues. Human HCS shows specific regions of homology with the BirA protein of E. coli and the presumptive biotin ligase of Paracoccus denitrificans. Several forms of HCS mRNA are generated by alternative splicing, and as a result, two mRNA molecules bear different putative translation initiation sites. A sequence upstream of the first translation initiation site encodes a peptide structurally similar to mitochondrial presequences, but it lacks an in-frame ATG codon to direct its translation. We anticipate that alternative splicing most likely mediates the mitochondrial versus cytoplasmic expression, although the elements required for directing the enzyme to the mitochondria remain to be confirmed.