Mitochondrial Quinone Reductases: Complex I

Giorgio Lenaz; Romana Fato; Alessandra Baracca; Maria Luisa Genova

Introduction

The NADH:quinone (Coenzyme Q, CoQ, ubiquinone) oxidoreductase (Complex I) is the most complicated enzyme of the respiratory chain in mitochondria and aerobic bacteria.1–3 Mitochondrial Complex I is a multisubunit enzyme that uses the energy associated to NADH oxidation by CoQ to pump hydrogen ions across the inner membrane, thus significantly contributing to the formation of an electrochemical proton gradient (ΔμH+) and consequently to the efficiency of the oxidative phosphorylation process. The number of protons pumped by Complex I is estimated to be 2 to 5 (probably 4) H+/2e−.4–8

In spite of recent improvements of our knowledge on both structural and functional properties of Complex I,9–11 the atomic structure and the detailed reaction mechanism of the enzyme are still unknown: it is the only enzyme of the membrane-bound respiratory chain to remain a “L shaped black box.”12 The reason for this lack of information is principally due to the complexity of this enzyme: in fact, the bovine enzyme consists of 46 different polypeptides13,14 with different prosthetic groups: an FMN, 8 to 9 Fe-S clusters and one or more molecules of quinones. The need to clarify the molecular mechanism of the enzyme complex is strongly supported by different research fields. Besides representing a major target of bioenergetics, since many neurodegenerative disorders as well as the aging process have been associated with Complex I deficiency,15–17 knowledge of the enzyme structure-function relationship is essential for better understanding of these physiological-pathological dysfunctions.

Therefore the availability of reliable methods allowing the study of the Complex I activity is very important to investigate the mechanism of electron transfer and proton translocation by the enzyme and to assess cell bioenergetic damage occurring in mitochondrial diseases and aging.

Assay of Redox Activities of Complex I

NADH-CoQ Reductase

Investigation of electron transfer in the complex along its redox groups is very difficult to perform. Moreover, isolation of Complex I in an active form is not an easy task,18 so the best way to assay its activity is to study the enzyme in situ, in mitochondrial membranes or in submitochondrial particles. From the functional point of view, Complex I activity can be isolated from the other respiratory complexes by the action of specific inhibitors such as antimycin A and mucidin for Complex III (acting respectively on center “i” and “o”) and cyanide for Complex IV. Myxothiazol should be avoided, since it also inhibits Complex I.19 Difficulties in assaying Complex I activity may arise from a limited permeability of its substrates; in particular, beef heart mitochondria, which are the choice material for Complex I studies, must be permabilized to NADH, and this may be achieved through freezing and thawing cycles (from 1 to 3 cycles). On the other hand, the complex uses Coenzyme Q10 as physiological electron acceptor; however, this molecule is too hydrophobic and cannot be used as exogenous substrate. For this reason Complex I activity is normally assayed by using short-chain analogs of CoQ10: the most widely used are CoQ1 (with only one isoprenoid unit in the side chain) and decylubiquinone (DB) with a 10-carbon-atom linear saturated side chain.20,21 This lack of a suitable assay method with endogenous substrates makes Complex I activity measurements considerably hampered. It has to be borne in mind that the activity of Complex I with these exogenous acceptors may be strongly underestimated.20,22,23

In Bovine Heart Mitochondria and Submitochondrial Particles

Beef heart mitochondria (BHM) are obtained by a large-scale procedure24 and submitochondrial particles (SMP) by sonic irradiation of frozen and thawed mitochondria.25 SMP obtained by this method are essentially broken membrane fragments26; alternatively, coupled closed particles (electron transfer particles or ETPH) are prepared by the method of Hansen and Smith27 or EDTA-particles by the method of Lee and Ernster.28

All preparations are kept frozen at −80° at a stock concentration ranging between 40 and 60 mg/ml of protein detected by the biuret method by Gornall et al.29 BHM are frozen and thawed two or three times before use, while SMP are used after thawing once: under these conditions the permeability barrier for NADH is completely lost, as demonstrated by the lack of further stimulation by detergents.

NADH-CoQ reductase is assayed essentially as described by Yagi30 and modified by Degli Esposti et al.19 and Estornell et al.21

Reagents

Buffer, 50 mM KCl, 10 mM Tris-HCl, 1 mM EDTA, pH 7.4

NADH, 30 mM freshly prepared solution in water

Antimycin A, 2 mM in ethanol

KCN, 2 mM in buffer solution

All quinones used are kept as 20 to 30 mM ethanol stock solutions

Rotenone, 2 mM in ethanol

Procedure

KCN and Antimycin A are added directly to the buffer to a final concentration of 2 mM and 2 μM, respectively; because of the alkaline hydrolysis of CN−, it is necessary to adjust the pH to 7.4 to 7.5 with HCl. KCN and Antimycin A are added to avoid the electron flow through the cytochrome system (complexes III and IV).

The reaction is started by the addition of 10 to 20 μg/ml of mitochondrial protein (either BHM or SMP) and different amounts of the quinone analogs used as external electron acceptor and is followed spectrophotometrically by the decrease of absorbance at 340 minus 380 nm of a saturating amount of NADH (75 μM) in a double wavelength spectrophotometer (Jasco V-550 equipped with a double wavelength accessory and a rapid mixing apparatus) with an extinction coefficient of 3.5 mM−1 cm−1. This extinction coefficient was directly calculated, taking in account that, in the double wavelength spectrophotometer used, the light transfer is due to an optical fiber device; the extinction coefficient may vary with other spectrophotometers.

We have used this method to study the kinetic parameters of different short chain quinone homologs such as CoQ0, CoQ1, CoQ2, CoQ3 and analogs having straight saturated chain such as 6-pentyl and 6-decylubiquinones (respectively PB and DB).21,31 The results obtained are listed in Table I.

As shown in the table, different quinones elicited different NADH-CoQ reductase activity, and this is principally due to the capability of quinone analogs to interact with the physiological active site of Complex I. The widely used method to test this capability is to measure the effect of a specific Complex I inhibitor such as rotenone: NADH-CoQ1, -CoQ2, -CoQ3, -DB, -PB are at least 90% sensitive to rotenone (2 μM), suggesting that all...