Masao Mochizuki

The low-spin heme of cytochrome c oxidase as the driving element of the proton-pumping process.

Mitochondrial cytochrome c oxidase plays an essential role in aerobic cellular respiration, reducing dioxygen to water in a process coupled with the pumping of protons across the mitochondrial inner membrane. An aspartate residue, Asp-51, located near the enzyme surface, undergoes a redox-coupled x-ray structural change, which is suggestive of a role for this residue in redox-driven proton pumping. However, functional or mechanistic evidence for the involvement of this residue in proton pumping has not yet been obtained. We report that the Asp-51 → Asn mutation of the bovine enzyme abolishes its proton-pumping function without impairment of the dioxygen reduction activity. Improved x-ray structures (at 1.8/1.9-Å resolution in the fully oxidized/reduced states) show that the net positive charge created upon oxidation of the low-spin heme of the enzyme drives the active proton transport from the interior of the mitochondria to Asp-51 across the enzyme via a water channel and a hydrogen-bond network, located in tandem, and that the enzyme reduction induces proton ejection from the aspartate to the mitochondrial exterior. A peptide bond in the hydrogen-bond network critically inhibits reverse proton transfer through the network. A redox-coupled change in the capacity of the water channel, induced by the hydroxyfarnesylethyl group of the low-spin heme, suggests that the channel functions as an effective proton-collecting region. Infrared results indicate that the conformation of Asp-51 is controlled only by the oxidation state of the low-spin heme. These results indicate that the low-spin heme drives the proton-pumping process.

Quantitative reevaluation of the redox active sites of crystalline bovine heart cytochrome c oxidase.

Approximately 30% of the iron contained in a bovine heart cytochrome c oxidase preparation was removed by crystallization, giving a molecular extinction coefficient 1.25–1.4 times higher than those reported thus far. Six electron equivalents provided by dithionite were required for complete reduction of the crystalline cytochrome c oxidase preparation. The fully reduced enzyme was oxidized with 4 oxidation equivalents provided by molecular oxygen, giving an absorption spectrum slightly, but significantly, different from that of the original fully oxidized form. Four electron equivalents were required for complete reduction of the O2-oxidized enzyme. The O2-oxidized form, when exposed to excess amounts of O2, was converted to the original oxidized form which required 6 electrons for complete reduction. A slow reduction of the O2-oxidized form without any external reductant added indicates the existence of internal electron donors for heme irons in the enzyme. These results suggest that the 2 extra oxidation equivalents in the original oxidized form, compared with the O2-oxidized form, are due to a bound peroxide produced by O2 and electrons from the internal donors, consistently with a peroxide at the O2 reduction site in the crystal structure of the enzyme (Yoshikawa, S., Shinzawa-Itoh, K., Nakashima, R., Yaono, R., Yamashita, E., Inoue, N., Yao, M., Fei, M. J., Peters Libeu, C., Mizushima, T., Yamaguchi, H., Tomizaki, T., and Tsukihara, T. (1998)Science 280, 1723–1729).

Structural studies on bovine heart cytochrome c oxidase

Among the X-ray structures of bovine heart cytochrome c oxidase (CcO), reported thus far, the highest resolution is 1.8Å. CcO includes 13 different protein subunits, 7 species of phospholipids, 7 species of triglycerides, 4 redox-active metal sites (Cu(A), heme a (Fe(a)), Cu(B), heme a(3) (Fe(a3))) and 3 redox-inactive metal sites (Mg(2+), Zn(2+) and Na(+)). The effects of various O(2) analogs on the X-ray structure suggest that O(2) molecules are transiently trapped at the Cu(B) site before binding to Fe(a3)(2+) to provide O(2)(-). This provides three possible electron transfer pathways from Cu(B), Fe(a3) and Tyr244 via a water molecule. These pathways facilitate non-sequential 3 electron reduction of the bound O(2)(-) to break the OO bond without releasing active oxygen species. Bovine heart CcO has a proton conducting pathway that includes a hydrogen-bond network and a water-channel which, in tandem, connect the positive side phase with the negative side phase. The hydrogen-bond network forms two additional hydrogen-bonds with the formyl and propionate groups of heme a. Thus, upon oxidation of heme a, the positive charge created on Fe(a) is readily delocalized to the heme peripheral groups to drive proton-transport through the hydrogen-bond network. A peptide bond in the hydrogen-bond network and a redox-coupled conformational change in the water channel are expected to effectively block reverse proton transfer through the H-pathway. These functions of the pathway have been confirmed by site-directed mutagenesis of bovine CcO expressed in HeLa cells.

The Mg2+-containing water cluster of mammalian cytochrome c oxidase collects four pumping proton equivalents in each catalytic cycle

Bovine heart cytochrome c oxidase (CcO) pumps four proton equivalents per catalytic cycle through the H-pathway, a proton-conducting pathway, which includes a hydrogen bond network and a water channel operating in tandem. Protons are transferred by H3O+ through the water channel from the N-side into the hydrogen bond network, where they are pumped to the P-side by electrostatic repulsion between protons and net positive charges created at heme a as a result of electron donation to O2 bound to heme a3. To block backward proton movement, the water channel remains closed after O2 binding until the sequential four-proton pumping process is complete. Thus, the hydrogen bond network must collect four proton equivalents before O2 binding. However, a region with the capacity to accept four proton equivalents was not discernable in the x-ray structures of the hydrogen bond network. The present x-ray structures of oxidized/reduced bovine CcO are improved from 1.8/1.9 to 1.5/1.6 Å resolution, increasing the structural information by 1.7/1.6 times and revealing that a large water cluster, which includes a Mg2+ ion, is linked to the H-pathway. The cluster contains enough proton acceptor groups to retain four proton equivalents. The redox-coupled x-ray structural changes in Glu198, which bridges the Mg2+ and CuA (the initial electron acceptor from cytochrome c) sites, suggest that the CuA-Glu198-Mg2+ system drives redox-coupled transfer of protons pooled in the water cluster to the H-pathway. Thus, these x-ray structures indicate that the Mg2+-containing water cluster is the crucial structural element providing the effective proton pumping in bovine CcO.

Effective Pumping Proton Collection Facilitated by a Copper Site (CuB) of Bovine Heart Cytochrome c Oxidase, Revealed by a Newly Developed Time-resolved Infrared System

Background: Cytochrome c oxidase reduces O2 coupled with proton pumping. Results: A newly developed time-resolved infrared system reveals transient conformational changes in the proton-pumping pathway upon CO binding to CuB in the O2 reduction site. Conclusion: CuB promotes proton collection and effective blockage of back-leak of pumping protons. Significance: These critical findings in bioenergetics stimulate the new infrared approach for mechanistic investigation of any other protein function. X-ray structural and mutational analyses have shown that bovine heart cytochrome c oxidase (CcO) pumps protons electrostatically through a hydrogen bond network using net positive charges created upon oxidation of a heme iron (located near the hydrogen bond network) for O2 reduction. Pumping protons are transferred by mobile water molecules from the negative side of the mitochondrial inner membrane through a water channel into the hydrogen bond network. For blockage of spontaneous proton back-leak, the water channel is closed upon O2 binding to the second heme (heme a3) after complete collection of the pumping protons in the hydrogen bond network. For elucidation of the structural bases for the mechanism of the proton collection and timely closure of the water channel, conformational dynamics after photolysis of CO (an O2 analog)-bound CcO was examined using a newly developed time-resolved infrared system feasible for accurate detection of a single C=O stretch band of α-helices of CcO in H2O medium. The present results indicate that migration of CO from heme a3 to CuB in the O2 reduction site induces an intermediate state in which a bulge conformation at Ser-382 in a transmembrane helix is eliminated to open the water channel. The structural changes suggest that, using a conformational relay system, including CuB, O2, heme a3, and two helix turns extending to Ser-382, CuB induces the conformational changes of the water channel that stimulate the proton collection, and senses complete proton loading into the hydrogen bond network to trigger the timely channel closure by O2 transfer from CuB to heme a3.

Proteomic analysis of autoantigens associated with systemic lupus erythematosus: Anti-aldolase A antibody as a potential marker of lupus nephritis.

To screen for autoantibodies associated with systemic lupus erythematosus (SLE), we used proteomic approaches combining 2‐D PAGE and Western blot analysis, followed by protein identification by LC‐MS/MS analysis, resulting in the identification of aldolase A as a novel autoantigen in SLE. ELISA showed the prevalence of anti‐aldolase A antibodies to be 29.3% in SLE, 8.2% in rheumatoid arthritis, 18.1% in polymyositis and absent in healthy controls. Furthermore, 43.4% of SLE patients suffering from nephritis showed anti‐aldolase A autoantibodies, which was significantly higher than the prevalence for those without nephritis (11.1%). In lupus nephritis, there are few reliable diagnostic methods, other than urinalysis. Therefore, these results indicate that autoantibodies against aldolase A may serve as an alternative clinical biomarker of SLE associated with nephritis.

X-ray structure of cyanide-bound bovine heart cytochrome c oxidase in the fully oxidized state at 2.0 Å resolution

The X-ray structure of cyanide-bound bovine heart cytochrome c oxidase in the fully oxidized state was determined at 2.0 Å resolution. The structure reveals that the peroxide that bridges the two metals in the fully oxidized state is replaced by a cyanide ion bound in a nearly symmetric end-on fashion without significantly changing the protein conformation outside the two metal sites.

Nanosecond Time-Resolved Infrared Basis for a Bulge of the Transmembrane Helix Between Hemes A and A3 to Facilitate Highly Efficient Proton Pumping by Bovine Heart Cytochrome C Oxidase

Recent X-ray structural analyses show that net positive charges on a heme iron site (heme a) of bovine cytochrome c oxidase, created upon electron donation to the O2-reduction site, electrostatically drives proton-pump through a hydrogen-bond network to the positive side of mitochondrial inner membrane [1, 2]. The four electron equivalents for complete reduction of O2 at the fully reduced O2-reduction site are transferred one at a time, each, coupled with pumping of one proton equivalent, giving four intermediate species, F, O, E and R (from P). X-ray structures of P, F, O and R suggested that the water channel which connects the negative side space with the hydrogen bond network is kept closed by Ser382 bulge of the trans-membrane-helix during the transfer of four electron equivalents for complete reduction of the bound O2. The closure blocks effectively proton back-leakage from the hydrogen-bond network.The structural basis for complete protonation of the hydrogen-bond network before the O2-binding prerequisite for the efficient energy transduction was explored using a newly developed nanosecond time-resolved infrared apparatus for aqueous protein system. A transient CO-binding to CuB, after Fea3-CO photolysis, was discovered to open the water-channel by eliminating the Ser382 bulge. The infrared and X-ray structural results suggest that, sensing protonation state of the hydrogen-bond network, a relay system including CuB, O2, Fea3 and two a-helix turns extending to Ser382 facilitates effective proton collection and timely water-channel closure by conformational changes in the Ser382-containing segment, thereby ensuring efficient energy transduction.[1] Muramoto K., et al (2010) Proc. Natl. Acad. Sci. USA 107: 7740-7745.[2] Yoshikawa S., et al (2011) Ann. Rev. Biophys. 40: 205-223.