Redox-linked proton transfer by cytochrome c oxidase

The respiratory chain is found in the inner mitochondrial membrane of higher organisms and in the plasma membrane of many bacteria. It consists of several membrane-spanning enzymes, which conserve the energy that is liberated from the degradation of food molecules as an electrochemical proton gradient across the membrane. The proton gradient can later be utilized by the cell for different energy requiring processes, e.g. ATP production, cellular motion or active transport of ions. The difference in proton concentration between the two sides of the membrane is a result of the translocation of protons by the enzymes of the respiratory chain, from the negatively charged (N-side) to the positively charged side (P-side) of the lipid bilayer, against the proton concentration gradient. The endergonic proton transfer is driven by the flow of electrons through the enzymes of the respiratory chain, from low redox-potential electron donors to acceptors of higher potential, and ultimately to oxygen. Cytochrome c oxidase is the last enzyme in the respiratory chain and catalyzes the reduction of dioxygen to water. The redox reaction is coupled to proton transport across the membrane by a yet unresolved mechanism. Cytochrome c oxidase has two proton-conducting pathways through which protons are taken up to the interior part of the enzyme from the N-side of the membrane. The K-pathway transfers merely substrate protons, which are consumed in the process of water formation at the catalytic site. The D-pathway transfers both substrate protons and protons that are pumped to the P-side of the membrane. This thesis focuses on the role of two conserved amino acids in proton translocation by cytochrome c oxidase, glutamate 278 and tryptophan 164. Glu278 is located at the end of the D-pathway and is thought to constitute the branching point for substrate and pumped protons. In this work, it was shown that although Glu278 has an important role in the proton transfer mechanism, its presence is not an obligatory requirement. Alternative structural solutions in the area around Glu278, much like the ones present in some distantly related heme-copper oxidases, could in the absence of Glu278 support the formation of a long hydrogen-bonded water chain through which proton transfer from the D-pathway to the catalytic site is possible. The other studied amino acid, Trp164, is hydrogen bonded to the ∆-propionate of heme a3 of the catalytic site. Mutation of this amino acid showed that it may be involved in regulation of proton access to a proton acceptor, a pump site, from which the proton later is expelled to the P-side of the membrane. The ion pair that is formed by the ∆-propionate of heme a3 and arginine 473 is likely to form a gate-like structure, which regulates proton mobility to the P-side of the membrane. The same gate may also be part of an exit path through which water molecules produced at the catalytically active site are removed towards the external side of the membrane. Time-resolved optical and electrometrical experiments with the Trp164 to phenylalanine mutant revealed a so far undetected step in the proton pumping mechanism. During the A to PR transition of the catalytic cycle, a proton is transferred from Glu278 to the pump site, located somewhere in the vicinity of the ∆-propionate of heme a3. A mechanism for proton pumping by cytochrome c oxidase is proposed on the basis of the presented results and the mechanism is discussed in relation to some relevant experimental data. A common proton pumping mechanism for all members of the heme-copper oxidase family is moreover considered.

Determinants of Coronary and Carotid Atherosclerosis in Finnish Patients with Clinically Suspected Coronary Artery Disease. A Quantitative Angiography and Ultrasound Study

Background. Cardiovascular disease (CVD) remains the most serious threat to life and health in industrialized countries. Atherosclerosis is the main underlying pathology associated with CVD, in particular coronary artery disease (CAD), ischaemic stroke, and peripheral arterial disease. Risk factors play an important role in initiating and accelerating the complex process of atherosclerosis. Most studies of risk factors have focused on the presence or absence of clinically defined CVD. Less is known about the determinants of the severity and extent of atherosclerosis in symptomatic patients. Aims. To clarify the association between coronary and carotid artery atherosclerosis, and to study the determinants associated with these abnormalities with special regard to novel cardiovascular risk factors. Subjects and methods. Quantitative coronary angiography (QCA) and B-mode ultrasound were used to assess coronary and carotid artery atherosclerosis in 108 patients with clinically suspected CAD referred for elective coronary angiography. To evaluate anatomic severity and extent of CAD, several QCA parameters were incorporated into indexes. These measurements reflected CAD severity, extent, and overall atheroma burden and were calculated for the entire coronary tree and separately for different coronary segments (i.e., left main, proximal, mid, and distal segments). Maximum and mean intima-media thickness (IMT) values of carotid arteries were measured and expressed as mean aggregate values. Furthermore, the study design included extensive fasting blood samples, oral glucose tolerance test, and an oral fat-load test to be performed in each participant. Results. Maximum and mean IMT values were significantly correlated with CAD severity, extent, and atheroma burden. There was heterogeneity in associations between IMT and CAD indexes according to anatomical location of CAD. Maximum and mean IMT values, respectively, were correlated with QCA indexes for mid and distal segments but not with the proximal segments of coronary vessels. The values of paraoxonase-1 (PON1) activity and concentration, respectively, were lower in subjects with significant CAD and there was a significant relationship between PON1 activity and concentration and coronary atherosclerosis assessed by QCA. PON1 activity was a significant determinant of severity of CAD independently of HDL cholesterol. Neither PON1 activity nor concentration was associated with carotid IMT. The concentration of triglycerides (TGs), triglyceride-rich lipoproteins (TRLs), oxidized LDL (oxLDL), and the cholesterol content of remnant lipoprotein particle (RLP-C) were significantly increased at 6 hours after intake of an oral fatty meal as compared with fasting values. The mean peak size of LDL remained unchanged 6 hours after the test meal. The correlations between total TGs, TRLs, and RLP-C in fasting and postprandial state were highly significant. RLP-C correlated with oxLDL both in fasting and in fed state and inversely with LDL size. In multivariate analysis oxLDL was a determinant of severity and extent of CAD. Neither total TGs, TRLs, oxLDL, nor LDL size were linked to carotid atherosclerosis. Insulin resistance (IR) was associated with an increased severity and extent of coronary atherosclerosis and seemed to be a stronger predictor of coronary atherosclerosis in the distal parts of the coronary tree than in the proximal and mid parts. In the multivariate analysis IR was a significant predictor of the severity of CAD. IR did not correlate with carotid IMT. Maximum and mean carotid IMT were higher in patients with the apoE4 phenotype compared with subjects with the apoE3 phenotype. Likewise, patients with the apoE4 phenotype had a more severe and extensive CAD than individuals with the apoE3 phenotype. Conclusions. 1) There is an association between carotid IMT and the severity and extent of CAD. Carotid IMT seems to be a weaker predictor of coronary atherosclerosis in the proximal parts of the coronary tree than in the mid and distal parts. 2) PON1 activity has an important role in the pathogenesis of coronary atherosclerosis. More importantly, the study illustrates how the protective role of HDL could be modulated by its components such that equivalent serum concentrations of HDL cholesterol may not equate with an equivalent, potential protective capacity. 3) RLP-C in the fasting state is a good marker of postprandial TRLs. Circulating oxLDL increases in CAD patients postprandially. The highly significant positive correlation between postprandial TRLs and postprandial oxLDL suggests that the postprandial state creates oxidative stress. Our findings emphasize the fundamental role of LDL oxidation in the development of atherosclerosis even after inclusion of conventional CAD risk factors. 4) Disturbances in glucose metabolism are crucial in the pathogenesis of coronary atherosclerosis. In fact, subjects with IR are comparable with diabetic subjects in terms of severity and extent of CAD. 5) ApoE polymorphism is involved in the susceptibility to both carotid and coronary atherosclerosis.

Oriin seminaaliplasman matriksimetalloproteinaasit

Siirto- ja pakastesiemennysten lisääntyessä tammojen hedelmällisyystulosten parantuminen on hevoskasvatuksen taloudellisuuden kannalta merkittävää. Oriitten spermoissa on eroja sekä siirto- että pakastekestävyydessä. Pelkällä orivalinnalla ei voida kuitenkaan vaikuttaa hedelmällisyystuloksiin, sillä yleensä valinta painottuu suorituskykyyn. Siittiöiden lisäksi seminaaliplasmalla on havaittu vaikutuksia siirto- ja pakastuskestävyyteen. Seminaaliplasma koostuu useista erilaisista biologisista komponenteista, joista matriksimetalloproteinaasit (MMP) ovat yksi. Ne ovat proteiiniperhe, johon kuluu useita jäseniä. MMP:t kykenevät hajottamaan muun muassa solun ulkoisia tukirakenteita sekä tyvikalvoa erilaisissa fysiologisissa ja patologisissa tiloissa. Matriksimetalloproteinaaseja on löydetty useista kudoksista ja myös seminaaliplasmasta. Työn tutkimusosuudessa haluttiin selvittää MMP-pitoisuuksia, niiden vaihteluita oriitten välillä ja mahdollisia vaikutuksia hedelmällisyystuloksiin. Seminaaliplasmanäytteitä tutkittiin yhteensä 43 oriista. Näytteet oli kerätty astutuskaudella 2006 erirotuisilta ja -ikäisiltä hevosilta sekä kahdelta ponilta. Keräysvaiheessa näytteet jaoteltiin 1-4 eri fraktioon. Jokaisesta näytteestä tutkittiin MMP:t zymografian avulla. Seminaaliplasmanäytteiden lisäksi oriilta kerättiin hedelmällisyystietoja siittoloista sekä Suomen raviurheilun ja hevoskasvatuksen keskusjärjestöltä (Suomen Hippos ry). Kaikki tulokset taulukoitiin ja laskettiin aktiiviselle (akt-MMP-2) ja pro-MMP-2:lle sekä kokonais-MMP-9:lle (tot-MMP-9) siittiörikkaassa (SR) ja siittiököyhässä fraktiossa (SK) sekä kokonaisejakulaatissa (KE): keskiarvot, keskihajonnat, mediaanit sekä maksimi- ja minimiarvot. Spearmanin järjestyskorrelaatiokertoimet laskettiin tammojen ensimmäiseen kiimaan tiinehtymisen ja eri MMP-pitoisuuksien välille SR:ssa ja KE:ssa. Oriilla oli havaittavia pitoisuuksia pro- ja akt-MMP-2:ta sekä tot-MMP-9:ää. MMP-pitoisuudet olivat suurimmat SR:ssa. Suurimpia olivat pro-MMP-2:n pitoisuudet ja orikohtaiset erot olivat siinä pieniä. Saadut tulokset vastasivat odotuksia, sillä miesten seminaaliplasmatutkimusten tulokset ovat samansuuntaisia. Eri MMP-pitoisuuksilla ei havaittu korrelaatiota tammojen tiinehtymiseen kanssa. Aineiston pienen koon takia sattumalla voi olla suuri vaikutus tuloksiin. Myös keräysvuodenaika ja yksilön ejakulaation koostumusvaihtelut saattavat vaikuttaa seminaaliplasman MMP-pitoisuuksiin, sillä sen useissa ominaisuuksissa tapahtuu muutoksia näiden muuttujien mukaan. Tulokset ovat suuntaa antavia ja toimivat apuna jatkotutkimuksia suunniteltaessa.