Photoaffinity Labeling of Respiratory Complex I in Bovine Heart Submitochondrial Particles by Photoreactive [125I] amilorides

Abstract

The architecture of quinone/inhibitor-access channel in proton-translocating NADH-quinone oxidoreductase (respiratory complex I) was modeled by X-ray crystallography and cryo-EM, however, it remains debatable whether the channel model reflects the physiologically relevant state present throughout the catalytic cycle. Using photoreactive [¹²⁵I]amilorides, we demonstrated that amiloride-type inhibitors bind to the interfacial region of multiple subunits (49-kDa, ND1, PSST, and 39-kDa subunits), which is difficult to reconcile with the current channel model. This report describes the procedures for photoaffinity labeling of bovine submitochondrial particles by photoreactive [¹²⁵I]amilorides. The protocol could be widely applicable for the characterization of various biologically active compounds, whose target protein remains to be identified or characterized.

Keywords: Amilorides; Bioenergetics; Chemical biology; Enzyme inhibitor; Mitochondria; NADH-quinone oxidoreductase; Photoaffinity labeling; Respiratory complex I.

Figure 1.. Schematic presentation of photoaffinity labeling of bovine heart SMPs by [¹²⁵I]amilorides

Figure 2.. Purification of photoreactive [¹²⁵I]PRA5 by reverse phase HPLC.

The mixture containing crude [¹²⁵I]PRA5 was applied to a C18 column. The salts were removed by rinsing the column with 10% methanol/0.01% aqueous TFA (0-10 min), then [¹²⁵I]PRA5 was eluted with an isocratic 90% methanol/0.01% aqueous TFA (10-20 min). The elute was fractionated every 30 s, and 2 µl aliquot from each fraction was transferred to RIA tube and the radioactivity was measured using γ-counter (retention time; 10-17.5 min). A. Radioactivity distribution in HPLC chromatogram. The radiochemical yield was approximately 70% from the initial [¹²⁵I]NaI. B. TLC analysis of the collected fractions. The samples (1 µl) were analyzed on a TLC plate using 10% methanol/chloroform as a mobile phase. The plate was exposed on an imaging plate for 1 h and analyzed by Bio-imaging analyzed FLA-5100.

Figure 3.. A set-up for photoaffinity labeling of bovine SMPs.

The 1.5 ml Eppendorf tubes containing SMPs are placed with open lids on ice. They are UV-irradiated for 10 min at the distance of approximately 10 cm from the light source. For efficient labeling, the volume of the sample should be 100-200 µl/tube.

Figure 4.. Preparation of the second dimensional gel for dSDS-PAGE.

A. Excise the first dimensional gel strip. B. Carefully place the gel strip to a 15 ml falcon tube containing an acidic buffer. Incubate the gel for 30 min at room temperature. C. Fix the strip between two glass plates. D. Pour the second dimensional acrylamide mixture, and overlay with Milli-Q H₂O. E. After polymerization of the acrylamide gel mixture, push down the gel strip to make the two gels stick together. F. Remove Milli-Q, then fill the gap with a spacer gel.

Figure 5.. Analysis of the proteins in SMPs labeled by [¹²⁵I]PRA5.

A. Separation of complex I by BN-PAGE. The SMPs labeled by [¹²⁵I]PRA5 were separated on a 4-16% BN gel. The complex I band was identified by activity stain by NADH/NBT system, as described elsewhere ( Murai et al., 2009 ; Shiraishi et al., 2012 ), and subjected to electroelution. Approximately 100 µg of SMPs proteins were loaded into each well. B. Resolution of complex I by dSDS-PAGE. The [¹²⁵I]PRA5-labeled complex I, purified by BN-PAGE and electroelution, was separated on a first dimensional 10% Schägger-type Tricine gel (10% T, 3% C containing 6.0 M urea), followed by second dimensional separation on a 16% Schagger-type gel (16% T, 3% C). The 2D gel was subjected to silver stain (left) and autoradiography (right). The labeling by [¹²⁵I]PRA5 provided two radioactive spots corresponding to the 49-kDa and PSST subunits, both of which comprise the quinone/inhibitor-binding pocket of complex I (Hirst 2013 and Sazanov 2015). The spots were identified by mass spectrometry and Western blotting ( Shiraishi et al., 2012 ). We note that the weak radioactivity was found in a protein spot of ADP/ATP carrier (AAC), which was co-purified with complex I. The gel images are the same as those used in Figure 7C of the reference ( Uno et al., 2019 ).