Role of cytochrome b5 reductase on the antioxidant function of coenzyme Q in the plasma membrane

Molec. Aspects Med. Vol. 18 (Supplement), pp. s7-~13, 1997 0 1997 Elsevier Science Ltd. All rights reserved Printed in Great Britain 0098-2997197 $32.00+0.00

ergamon

PII:

SOO98-2997(97)00015-O

Role of Cytochrome b5 Reductase on the Antioxidant Function of Coenzyme Q in the Plasma Membrane J. M. Villalba, Depattamento

F. Navarro, C. G6mez-Diaz,

de Siologia

A. Arroyo, R. I. Belle and P. Navas

Celular, Facultad de Ciencias, Universidad 74004Cdrdoba, Spain

de Cdrdoba,

Abstract-Cytochrome b5 reductase purified from liver plasma membrane reduces coenzyme Q (CoQ) in reconstituted liposomes in the absence of cytochrome b5. Both CoQ and its reductase are responsible for the reduction of the ascorbate free radical at the cell surface. Thus, NADH-CoQ reductase represents a partial reaction of NADH-AFR reductase in the plasma membrane. Cytochrome b5 reductase maintains CoQ and ascorbate in their reduced state to support antioxidations. Reduced CoQ prevents lipid peroxidation in liposomes and plasma membranes. Also, oxidized CoQ can prevent lipid peroxidations in the presence of cytochrome b5 reductase and NADH. Addition of CoQ to intact cells prevents serum withdrawal-induced lipid peroxidation and apoptosis. The prevention of apoptosis by CoQ is independent of the bcl-2 protein content in the cell. Antioxidants that act at the plasma membrane as CoQ and ascorbate would represent a first barrier to protect lipids from oxidative stress and subsequent apoptosis. Cytochrome b, reductase is then an enzyme leading this function at the plasma membrane. These data support the idea that when the plasma membrane barrier fails, bcl-2 protein would be required to prevent cell death. 0 1997 Elsevier Science Ltd Keywords:

apoptosis; ascorbate;

coenzyme Q; cytochrome b5 reductase;

lipid peroxidation

Introduction The

eukaryotic

plasma

membrane

contains

a redox

system

that

transfers

electrons

from intracellular donors to impermeable external oxidants. Transplasma membrane redox activity has been related to growth control and development (Crane et al.,

1985). Although there exists a general aggreement on NAD(P)H as the natural electron donor (Navas and Bur6n, 1990), the identification of natural acceptors for the transmembrane dehydrogenase is still a matter of controversy. In addition to oxygen s7

s8

J. M. Villalba et al.

and iron-containing compounds, the free radical (AFR), has been proposed

semioxidized as a natural

form of ascorbate, the ascorbate acceptor (Buren et al., 1987).

Ascorbate is a first order antioxidant that protects cellular components from free radical-induced damage, either by a direct quenching of various soluble free radicals, by scavenging lipid peroxidation-initiating radicals, or by reducing tocophero@ radicals to tocopherol (Frei, 1994). As humans and different animals can not synthesize this compound, the mechanisms to stabilize ascorbate available in the diet are of extreme importance. In fact, ascorbate in tissues is maintained primarily in the reduced state (Rose and Bode, 1993), clearly depending on the presence of cells (Minetti et al., 1992). Stabilization of ascorbate at the cell surface is a mechanism to maintain the antioxidant property of this vitamin and appears to be mediated by an enzyme system. Quenching of AFR at the cell surface (Pethig et al., 1985) shows similar properties to the ascorbate stabilization activity observed in z&r-o (RodriguezAguilera and Navas, 1994), but the nature of the enzymatic components responsible for this stabilization has to be elucidated. Coenzyme Q (CoQ) is a lipid-soluble antioxidant that mediates the electron transport in the plasma membrane (Sun et al., 1992), also including AFR as an oxidant (Villalba et al., 1995). Interaction of both ascorbate and CoQ with x-tocopherol as a mechanism of membrane lipids protection is well established, but there was no evidence of direct interaction between ascorbate and CoQ in membranes (Beyer, 1994). The participation of different antioxidants as components of the plasma membrane redox system is emphasized here. This electron transport system drives electrons from the cytosolic NADH to the extracellular ascorbate free radical through the cytochrome b5 reductase, cytochrome b, being not required. This electron transport leads to the maintenance of ascorbate and CoQ in their reduced forms and can thus membranes from peroxidation and prevent cells from undergoing protect programmed death induced at the plasma membrane.

Role of Cytochrome b5 Reductase and Ascorbate Stabilization

in CoGmediated

Electron Transport

CoQ participates as an intermediate carrier in plasma membrane-associated redox activities (Sun et al., 1992; Villalba et al., 1995). Requirement for CoQ distinguishes transplasma membrane redox activity from activities related to cis electron transport such as NADH-cytochrome c oxidoreductase. Extraction of CoQ with heptane from lyophylized plasma membranes inhibited NADH-AFR reductase and addition of CoQ restored the activity (Table 1). In contrast, NADH-cytochrome c oxidoreductase was not affected by the CoQ status. A CoQ reductase has been purified from pig liver plasma membranes (Villalba et al., 1995), and its internal sequence showed identity with bovine microsomal NADHfor cytochrome b5 reductase (Navarro et al., 1995). This enzyme is also responsible the mitochondrial outer membrane NADH-AFR reductase, that uses an outer membrane specific cytochrome 6, (Shirabe et al., 1995). Cytochrome b5 content in

Cytochrome Table

1.

b5 reductase

Role of CoQ on NADH-AFR

as antioxidant

reductase

s9

of purified plasma membranes

Sample

Activity

%

Control Extracted Reconstituted Supplemented

7.5 4.5 8.1 11.9

-40 +g +59

Lyophilized membranes from K-562 cells (control) were extracted with heptane (extracted). CoQ in heptane was added to both extracted membranes (reconstituted) and control membranes (supplemented) to a final concentration of 50 PM. Variations were calculated from the difference relative to control. Negative values mean inhibition and positive variations mean activation. Specific activities were nmoles/min/mg protein. S.D. I 10% (n = 4).

plasma other plasma Steck, can be

membrane is apparently very variable and always very low compared with 1980). Although NADH-cytochrome c reductase in membranes (Remacle, membrane must use cytochrome 6, reductase and cytochrome b, (Kant and 1972), less than 40% of cytochrome b5 incorporated into the plasma membrane reduced by the NADH-cytochrome b5 reductase (Remacle, 1980).

These findings suggest the hypothesis that plasma membrane NADH-AFR reductase might be composed of a NADH-cytochrome b5 reductase functioning as CoQ reductase and a still unknown enzyme system that utilizes CoQ hydroquinone to reduce extracellular AFR, that results in ascorbate stabilization. In

the

short-term,

cells

decrease

the

oxidation

rate

of

ascorbate

and

increasing

NADH results in a further reduction of the autoxidation rate (Alcain et al., 1991). These results have been interpreted in most cases as a regeneration of most likely by transmembrane NADH-AFR reductase extracellular ascorbate, (Rodriguez-Aguilera and Navas, 1994). In IX562 cells, incorporation of NADHcytochrome b5 reductase increases ascorbate stabilization by whole cells and the on CoQ (Gomez-Diaz et al., 1996). The quenching of process is dependent extracellular AFR by Ehrlich ascites cells has been demonstrated to occur and to depend on the cell viablity, surface charge and cell surface thiol groups (Pethig et al., 1985). intracellular

Thus, plasma membrane cytochrome b5 reductase is able to mediate the regeneration of ascorbate in a manner apparently independent of cytochrome b5. AFR reacts with reduced cytochrome bs reductase but no reaction was observed with cytochrome b5 measured by pulse radiolysis. The rate of AFR-induced NADH oxidation by cytochrome b5 reductase was about 30 times lower than that of NADH-AFR reductase. Thus, this latter activity must have an active site structure that accepts the physiological substrate APR, and is probably deficient in cytochrome b5 reductase (Kobayashi et al., 1991).

Protection

Against Oxidative

Stress and Programmed

Cell Death

Cells protect themselves against oxidative damage either by enzymes that eliminate oxygen active species or breaking the free radical chain reaction by small molecules as ascorbate, tocopherols and CoQ. CoQ hydroquinone has been proposed to have a role

SlO

J. M. Villalba et al.

as a free radical chain breaking antioxidant most likely due to its capacity to regenerate a-tocopherol, although the direct scavenging of peroxyl radicals by CoQ hydroquinone has been also considered (Kagan et al., 1996). CoQ participates in the prevention of peroxidations in reconstituted egg phosphatidylcholine liposomes (Beyer, 1994), the reduced form, ubiquinol, being the most effective. In addition to the inner mitochondrial CoQ reductase, other systems may be operative for CoQ reduction in endomembranes, plasma membrane and the extracellular fluid (Schultz et al., 1996). A4mong the different systems so far described, the cytosolic enzyme DT-diaphorase and the membrane-bound enzymes cytochrome P4.50 and -b5 reductases are possibly the most studied examples of extramitochondrial CoQ reductases (Nakamura and Hayashi, 1994). The putative role of P450 and b5 reductases in the maintenance of cellular levels of hydroquinones has been criticized because of the formation of the intermediate semiquinone that, under certain circumstances, may lead to the generation of superoxide (Nakamura and Hayashi, 1994). However, the observed prooxidant or antioxidant actions of hydroquinones and semiquinones are largely dependent on the conditions used (Schultz et al., 1996). Also, results obtained with small, hydrophylic quinones cannot be readily applied to naturally occurring quinones with long isoprenoid side chains. In reconstituted systems, CoQ hydroquinone can be regenerated using NADH by the plasma membrane cytochrome b5 reductase (Navarro et al., 1995). Cytochrome b5 reductase inhibits lipid peroxidation initiated by azo compounds in the presence of NADH and CoQ (Fig. 1). Also, purified cytochrome P450 reductase was found to reduce tocopheroxyl radical in the presence of ubiquinones in a NADPH-dependent process (Goldman et al., 1993). Transplasma

membrane NADH-AFR reductase, that requires CoQ and cytochrome reduces the AFR to regenerate ascorbate, an antioxidant molecule that can also reduce the tocopheroxyl radical (Beyer, 1994). Thus, in addition to other quinone reductases, a role for cytochrome b5 reductase may be the maintenance of appropriate levels of antioxidants in the plasma membrane and this enzyme will probably be recognized as basic for membrane protection.

b5 reductase,

Diverse agents that induce an oxidative stress activate a cell death program (Vaux and Strasser, 1996). Different mechanisms involved in the protection of cells to prevent the programmed cell death, as the bcl-2 family of proteins, apparently function through an antioxidant pathway (Hockenbery et al., 1993). Although the programmed cell death is not only triggered by reactive oxygen species, there are evident changes in the intracellular redox equilibrium in cells undergoing apoptosis (Slater et al., 1996). Serum withdrawal from culture media represents the removal of growth factors required for cells to survive (Ishizaki et al., 1995). In this case, a death program is induced to some extent. HL-60 cells were moderately resistant to serum removal

Cytochrome

(20% apoptotic cells), which do not contain death was partially a-tocopherol.

b5 reductase

sl 1

as antioxidant

most likely because they express bcl-2. However, this protein, were more sensitive (60% apoptotic prevented by antioxidants such as ascorbate,

Daudi cells, cells). Cell CoQ and

Conclusions Cells require different mechanisms to protect membranes from oxidative stress to maintain their structure and function. In the plasma membrane, this mechanism is represented by CoQ and a-tocopherol inside the lipid bilayer, and ascorbate in the interphase. The isolation and characterization of a plasma membrane dehydrogenase that drives electrons to the AFR through the CoQ supports the hypothesis of a complex transmembrane electron transport. Oxidative stress initiated at the plasma membrane leads the cells to a programmed death that can be prevented by intracellular mechanisms as the bcl-2 family of proteins. When mild damage occuring at the plasma membrane can be repaired by intrinsic antioxidants, the intracellular pathway appears unnecessary.

Relative fluorescence

(bar =I0 %)

I Smin

Fig.

1. Protection

of liposomal

liposomes

(10 mgiml)

incubated

with

peroxidation

were

cytochrome

peroxidation

b5 reductase

was followed as a decrease

and p-hydroxymercuribenzoate

by cytochrome

loaded with 5 PM parinaric (5 pg) purified in fluorescence

(PHMB) emission

from

Egg phosphatidylcholine

excitation

was 413

with 50 PM CoQ and

pig liver plasma

of parinaric

at 100 PM. The wavelenght

b5 reductase.

acid, reconstituted

nm.

acid. NADH wavelength

membrane.

Lipid

was used at 30 PM was 324 nm and the

J. M. Villalba et a/.

s12

Acknowledgements This

work was supported

by Spanish

DGICYT

grant

no. PB95-0560-A.

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