Reactive oxygen species generation by reverse electron transfer at mitochondrial complex I under simulated early reperfusion conditions.

Ischemic tissues accumulate succinate, which is rapidly oxidized upon reperfusion, driving a burst of mitochondrial reactive oxygen species (ROS) generation that triggers cell death. In isolated mitochondria with succinate as the sole metabolic substrate under non-phosphorylating conditions, 90 % of ROS generation is from reverse electron transfer (RET) at the Q site of respiratory complex I (Cx-I). Together, these observations suggest Cx-I RET is the source of pathologic ROS in reperfusion injury. However, numerous factors present in early reperfusion may impact Cx-I RET, including: (i) High [NADH]; (ii) High [lactate]; (iii) Mildly acidic pH; (iv) Defined ATP/ADP ratios; (v) Presence of the nucleosides adenosine and inosine; and (vi) Defined free [Ca2+]. Herein, experiments with mouse cardiac mitochondria revealed that under simulated early reperfusion conditions including these factors, total mitochondrial ROS generation was only 56 ± 17 % of that seen with succinate alone (mean ± 95 % confidence intervals). Of this ROS, only 52 ± 20 % was assignable to Cx-I RET. A further 14 ± 7 % could be assigned to complex III, with the remainder (34 ± 11 %) likely originating from other ROS sources upstream of the Cx-I Q site. Together, these data suggest the relative contribution of Cx-I RET ROS to reperfusion injury may be overestimated, and other ROS sources may contribute a significant fraction of ROS in early reperfusion.

Reactive Oxygen Species Generation by Reverse Electron Transfer at Mitochondrial Complex I Under Simulated Early Reperfusion Conditions.

Ischemic tissues accumulate succinate, which is rapidly oxidized upon reperfusion, driving a burst of mitochondrial reactive oxygen species (ROS) generation that triggers cell death. In isolated mitochondria with succinate as the sole metabolic substrate under non-phosphorylating conditions, 90% of ROS generation is from reverse electron transfer (RET) at the Q site of respiratory complex I (Cx-I). Together, these observations suggest Cx-I RET is the source of pathologic ROS in reperfusion injury. However, numerous factors present in early reperfusion may impact Cx-I RET, including: (i) High [NADH]; (ii) High [lactate]; (iii) Mildly acidic pH; (iv) Defined ATP/ADP ratios; (v) Presence of the nucleosides adenosine and inosine; and (vi) Defined free [Ca2+]. Herein, experiments with mouse cardiac mitochondria revealed that under simulated early reperfusion conditions including these factors, overall mitochondrial ROS generation was only 56% of that seen with succinate alone, and only 52% of this ROS was assignable to Cx-I RET. The residual non-RET ROS could be partially assigned to complex III (Cx-III) with the remainder likely originating from other ROS sources upstream of the Cx-I Q site. Together, these data suggest the relative contribution of Cx-I RET ROS to reperfusion injury may be overestimated, and other ROS sources may contribute a significant fraction of ROS in early reperfusion.

D-to-E and T19V Variants of the pH-Low Insertion Peptide and Their Doxorubicin Conjugates Interact with Membrane at Higher pH Ranges Than WT.

To improve targeted cargo delivery to cancer cells, pH-Low Insertion Peptide (pHLIP) variants were developed to interact with the membrane at pH values higher than those of the WT. The Asp-to-Glu variants aim to increase side chain pKa without disturbing the sequence of protonations that underpin membrane insertion. The Thr19 variants represent efforts to perturb the critical Pro20 residue. To study the effect of cargo on pHLIP insertion, doxorubicin (Dox), a fluorescent antineoplastic drug, was conjugated to selected variants near the inserting C-terminus. Variants and conjugates were characterized on a POPC membrane using Trp and Dox fluorescence methods to define the entire pH range of insertion (pHinitial-pHfinal). Compared to WT with a pHi-pHf range of 6.7-5.6, D25E-D31E-D33E, D14E-D25E-D31E-D33E, and T19V-D25E variants demonstrated higher pHi-pHf ranges of 7.3-6.1, 7.3-6.3, and 8.2-5.4, respectively. The addition of Dox expanded the pHi-pHf range, mainly by shifting pHi to higher pH values (e.g., WT pHLIP-Dox has a pHi-pHf range of 7.7-5.2). Despite the low Hill coefficient observed for the conjugates, D14E-D25E-D31E-D33E pHLIP-Dox completed insertion by a pHf of 5.7. However, the Dox cargo remained in the hydrophobic membrane interior after pHLIP insertion, which may impede drug release. Finally, a logistic function can describe pHLIP insertion as a peripheral-to-TM (start-to-finish) two-state transition; wherever possible, we discuss data deviating from such sigmoidal fitting in support of the idea that pH-specific intermediate states distinct from the initial peripheral state and the final TM state exist at intervening pH values.