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First published online June 11, 2007
Journal of Experimental Biology 210, 2082-2090 (2007)
Published by The Company of Biologists 2007
doi: 10.1242/jeb.003947

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Myoglobin-enhanced oxygen delivery to isolated cardiac mitochondria

Jonathan B. Wittenberg and Beatrice A. Wittenberg

Department of Physiology and Biophysics, Albert Einstein College of Medicine, Bronx, NY 10461, USA

View larger version (5K): [in this window] [in a new window]   Fig. 1. Oxygen consumption reported by myoglobin deoxygenation monitored at 625 nm is determined in mitochondrial suspensions containing myoglobin and limiting amounts of ADP. ADP is exhausted at the arrows. Myoglobin, 500 µmol l–1. A, 500 µmol l–1 ADP; B, 1000 µmol l–1 ADP; C, 2000 µmol l–1 ADP. Initial rates are 200 mol O2 (mol cytochrome aa3)–1 min–1. P/O ratios are: 2.3, 2.4 and 2.4, respectively, with respiratory control indices of 3.1–3.4. The P/O ratio of this preparation of mitochondria, determined polarographically, was 2.4 with RCI>6. This shows that mitochondrial oxygen uptake is tightly coupled to phosphorylation when the mitochondria are consuming myoglobin-bound oxygen. AU, absorbance units.

View larger version (5K): [in this window] [in a new window]   Fig. 2. Oxygen pressure in a suspension of mitochondria reported polarographically as a function of time (1 torr=0.133 kPa). ADP (500 nmol) is added at the arrows. A, myoglobin absent; B, 500 µmol l–1 oxymyoglobin. Average oxygen uptake in the presence of adequate ADP is 193 and 157 mol O2 (mol cytochrome aa3)–1 min–1 in the absence and presence of myoglobin, respectively. P/O ratios are 2.21 and 2.20. The concentration of myoglobin used here exceeds the volume-average concentration in pigeon heart, 209 µmol l–1 (Schuder et al., 1979). These results show that myoglobin in the solution does not affect the respiratory parameters of isolated mitochondria.

View larger version (9K): [in this window] [in a new window]   Fig. 3. Myoglobin and leghemoglobin deoxygenation as functions of time in hemoglobin-containing suspensions of mitochondria. Initially the hemoglobins are fully oxygenated. Deoxygenation is down. (A) Myoglobin, 50 µmol l–1; mitochondria (as cytochrome aa3) 170 nmol l–1, 10 mm light path. (A, inset) Myoglobin 500 µmol l–1; mitochondria (as cytochrome aa3) 700 nmol l–1, 2 mm light path. (B) Leghemoglobin c, 50 µmol l–1; mitochondria (as cytochrome aa3) 65 nmol l–1, 10 mm light path. The broken lines are drawn at a tangent to the nearly linear portions of the curves. The arrows indicate the points at which the rates of mitochondrial oxygen uptakes are half that during the near-linear portion of the progress curves. The quasi-steady state oxygen uptakes calculated from the slopes of the broken lines are 204 and 358 mol O2 (mol cytochrome aa3)–1 min–1 for myoglobin and leghemoglobin, respectively. The oxygen pressure at half-maximal oxygen uptake is found to be the same for two proteins for which affinities differ 10-fold (see Table 2). This shows that the PO2 for half-maximal mitochondrial oxygen uptake is not related to the oxygen affinity of the hemeprotein supplying oxygen. AU, absorbance units.

View larger version (6K): [in this window] [in a new window]   Fig. 4. Mitochondrial oxygen uptake as functions of myoglobin or leghemoglobin concentration. Closed symbols, myoglobin; open symbols, leghemoglobin c. Different symbols represent different experiments. With the exception of the two lowest points, oxygen uptake rates are those reported spectrophotometrically by the near-steady-state rates of deoxygenation of myoglobin or leghemoglobin, as in Fig. 3. Each point is the average of measurements on seven (myoglobin) or two (leghemoglobin) preparations of isolated mitochondria. The two lowest points were determined using trace concentrations of myoglobin or leghemoglobin as a reporter of oxygen pressure. Oxygen uptakes are normalized to a plateau value determined by the polarographically determined specific activity. The ratios of the rates of oxygen uptake at the plateau of this figure to those determined polarographically were 0.91, 0.77 and 1.30 in experiments using myoglobin and 0.76, 0.96 and 0.92 in experiments using leghemoglobin.

View larger version (5K): [in this window] [in a new window]   Fig. 5. Mitochondrial oxygen uptake as a function of myoglobin concentration using NADH (1 mmol l–1) as the sole substrate. For this experiment mitochondria were prepared by a less stringent procedure, making the mitochondria able to use exogenous NADH. Polarographically determined specific oxygen uptakes of this mitochondrial preparation were 134 and 412 mol O2 (mol cytochrome aa3)–1 min–1 using glutamate/malate or NADH (1 mmol l–1), respectively, as substrate. The latter may be compared to the rate exhibited by mitochondria fully uncoupled by exposure to carbonyl cyanide m-chlorophenylhydrazone (CCCP), 700 mol O2 (mol cytochrome aa3)–1 min–1.