Oliver-Matthias H. Richter

Cytochrome c Oxidase (Heme aa3) from Paracoccus denitrificans: Analysis of Mutations in Putative Proton Channels of Subunit I

One of the challenging features of energy-transducing terminal oxidases, like the aa3 cytochrome c oxidase of Paracoccus denitrificans, is the translocation of protons across the cytoplasmic membrane, which is coupled to the transfer of electrons to oxygen. As a prerequisite for a more advanced examination of the enzymatic properties, several amino acid residues, selected on the basis of recent three-dimensional structure determinations, were exchanged in subunit I of the Paracoccus enzyme by site-directed mutagenesis. The properties of the mutated oxidases were analyzed by different methods to elucidate whether they are involved in the coupled and coordinated transfer of protons via two different pathways either to the site of oxygen reduction or through the enzyme from the cytoplasm to the periplasmic side.

The structure of porin from Paracoccus denitrificans at 3.1 A resolution

© 1997 Federation of European Biochemical Societies.

An oxo-ferryl tryptophan radical catalytic intermediate in cytochrome c and quinol oxidases trapped by microsecond freeze-hyperquenching (MHQ)

The pre‐steady state reaction kinetics of the reduction of molecular oxygen catalyzed by fully reduced cytochrome oxidase from Escherichia coli and Paracoccus denitrificans were studied using the newly developed microsecond freeze‐hyperquenching mixing‐and‐sampling technique. Reaction samples are prepared 60 and 200 μs after direct mixing of dioxygen with enzyme. Analysis of the reaction samples by low temperature UV–Vis spectroscopy indicates that both enzymes are trapped in the PM state. EPR spectroscopy revealed the formation of a mixture of two radicals in both enzymes. Based on its apparent g‐value and lineshape, one of these radicals is assigned to a weakly magnetically coupled oxo‐ferryl tryptophan cation radical. Implications for the catalytic mechanism of cytochrome oxidases are discussed.

BIOCHEMICAL AND SPECTROSCOPIC PROPERTIES OF THE FOUR-SUBUNIT QUINOL OXIDASE (CYTOCHROME BA3) FROM PARACOCCUS DENITRIFICANS

The ba3 quinol oxidase from Paracoccus denitrificans has been purified by a new protocol leading to significantly higher yields than previously reported (Richter et al. (1994) J. Biol. Chem. 269, 23079-23086). In an SDS PAG an additional protein band compared with the previous preparation appears, which can be identified as the major form of subunit II. All protein bands can be assigned to genes of the qox operon by N-terminal sequencing, indicating that the oxidase consists of four subunits. In addition to one heme A, one heme B, and one copper atom, the preparation contains two ubiquinone molecules per enzyme. The oxidase is further characterized by electron paramagnetic resonance (EPR), circular dichroism (CD) and magnetic circular dichroism (MCD) spectroscopy.

Allosteric interactions and proton conducting pathways in proton pumping aa3 oxidases: Heme a as a key coupling element ☆

In this paper allosteric interactions in protonmotive heme aa(3) terminal oxidases of the respiratory chain are dealt with. The different lines of evidence supporting the key role of H(+)/e(-) coupling (redox Bohr effect) at the low spin heme a in the proton pump of the bovine oxidase are summarized. Results are presented showing that the I-R54M mutation in P. denitrificans aa(3) oxidase, which decreases by more than 200mV the E(m) of heme a, inhibits proton pumping. Mutational amino acid replacement in proton channels, at the negative (N) side of membrane-inserted prokaryotic aa(3) oxidases, as well as Zn(2+) binding at this site in the bovine oxidase, uncouples proton pumping. This effect appears to result from alteration of the structural/functional device, closer to the positive, opposite (P) surface, which separates pumped protons from those consumed in the reduction of O(2) to 2 H(2)O.

Expression of the Escherichia coli cyo operon in Paracoccus denitrificans results in a fully active quinol oxidase of unexpected heme composition

The cyo operon coding for the membrane‐bound bo 3‐type quinol oxidase of Escherichia coli has been expressed in a Paracoccus denitrificans strain deleted in its endogenous ba 3 quinol oxidase. Using the P. denitrificans qox promoter, the His tagged protein complex is synthesized to a level comparable to that in E. coli and the enzyme purified in a single step on a metal‐chelating column. Whereas the activity of the isolated complex matches that of the oxidase purified directly from E. coli, the heterologously expressed oxidase does not show the characteristic heme composition but now carries heme a in its binuclear site.

The Protein Effect in the Structure of Two Ferryl-Oxo Intermediates at the Same Oxidation Level in the Heme Copper Binuclear Center of Cytochrome c Oxidase

Background: Understanding the coupling of O2 reduction to proton pumping by CcO requires detection of reaction intermediates. Results: We have detected two oxoferryl intermediates at the PM oxidation state. Conclusion: The H-bonding properties of the proximal heme a3 His ligand control the strength of the oxoferryl species. Significance: The role of His-411, Thr-389, Gly-386, and Asp-399 residues in the proton pumping P→F transition is outlined. Identification of the intermediates and determination of their structures in the reduction of dioxygen to water by cytochrome c oxidase (CcO) are particularly important to understanding both O2 activation and proton pumping by the enzyme. In this work, we report the products of the rapid reaction of O2 with the mixed valence form (CuA2+, heme a3+, heme a32+-CuB1+) of the enzyme. The resonance Raman results show the formation of two ferryl-oxo species with characteristic Fe(IV)=O stretching modes at 790 and 804 cm−1 at the peroxy oxidation level (PM). Density functional theory calculations show that the protein environment of the proximal H-bonded His-411 determines the strength of the distal Fe(IV)=O bond. In contrast to previous proposals, the PM intermediate is also formed in the reaction of Y167F with O2. These results suggest that in the fully reduced enzyme, the proton pumping νFe(IV)=O = 804 cm−1 to νFe(IV)=O = 790 cm−1 transition (P→F, where P is peroxy and F is ferryl) is triggered not only by electron transfer from heme a to heme a3 but also by the formation of the H-bonded form of the His-411-Fe(IV)=O conformer in the proximal site of heme a3. The implications of these results with respect to the role of an O=Fe(IV)-His-411-H-bonded form to the ring A propionate of heme a3-Asp-399-H2O site and, thus, to the exit/output proton channel (H2O) pool during the proton pumping P→F transition are discussed. We propose that the environment proximal to the heme a3 controls the spectroscopic properties of the ferryl intermediates in cytochrome oxidases.

A Novel Heme a Insertion Factor Gene Cotranscribes with the Thermus thermophilus Cytochrome ba3 Oxidase Locus

Studying the biogenesis of the Thermus thermophilus cytochrome ba(3) oxidase, we analyze heme a cofactor insertion into this membrane protein complex. Only three proteins linked to oxidase maturation have been described for this extreme thermophile, and in particular, no evidence for a canonical Surf1 homologue, required for heme a insertion, is available from genome sequence data. Here, we characterize the product of an open reading frame, cbaX, in the operon encoding subunits of the ba(3)-type cytochrome c oxidase. CbaX shares no sequence identity with any known oxidase biogenesis factor, and CbaX homologues are found only in the Thermaceae group. In a series of cbaX deletion and complementation experiments, we demonstrate that the resulting ba(3) oxidase complexes, affinity purified via an internally inserted His tag located in subunit I, are severely affected in their enzymatic activities and heme compositions in both the low- and high-spin sites. Thus, CbaX displays typical features of a generic Surf1 factor essential for binding and positioning the heme a moiety for correct assembly into the protein scaffold of oxidase subunit I.

Chimeric Quinol Oxidases Expressed in Paracoccus Denitrificans

Quinol oxidases from both Escherichia coli (cytochrome bo 3) and Paracoccus denitrificans (ba 3) contain four different subunits supposedly sharing similar membrane topography, and are coded for in the cyo and qox operon, respectively. By deleting stopcodons in intercistronic regions, we have constructed different fusions between the P. denitrificans quinol oxidase subunits; all complexes are expressed in P. denitrificans and show enzymatic activities in membranes that are comparable to the unfused complex. Subsequently, chimeric complexes are constructed in which the subunit II gene (cyoA) replaces the endogenous qoxA; it is expressed either as an individual subunit or as a fused chimera, assembled with the other subunits of the P. denitrificans cytochrome ba 3 complex. Such heterologous enzymes retain a residual activity of 15–20% both in membranes and in the isolated complexes, which are purified in a two-step procedure by affinity chromatography using a strep-tag on subunit I. Spectral and protein chemical data further suggest a certain degree of instability of the mutated chimeric complexes. We conclude, however, that subunit II of E. coli basically interacts with the remaining subunits, notably subunit I, of the P. denitrificans ba 3 oxidase in the same way as the endogenous subunit, and sustains quinol oxidation and electron transfer activity to subunit I.