IUBMB
Life, 56(1): 7–11, January 2004
Review Article Reactions of Nitric Oxide with Copper Containing Oxidases; Cytochrome c Oxidase and Laccase Michael T. Wilson1 and Jaume Torres2 1
Department of Biological Sciences, University of Essex, Colchester CO4 3SQ, UK School of Biological Sciences, Nanyang Technological University, NTU 637616 Singapore
2
Summary The reactions of nitric oxide with copper containing oxidases such as cytochrome c oxidase and laccase are described and discussed in the present review. IUBMB Life, 56: 7–11, 2004 Keywords Nitric oxide; copper containing oxidases; cytochrome c oxidase; laccase; NO redox reactions.
INTRODUCTION Nitric oxide (NO) has a complex solution chemistry that involves interactions with metal ions, comprising both binding and redox reactions, and reactions with molecules such as dioxygen and reactive oxygen species such as superoxide (1). Secondary reactions lead to the chemical modification of biological molecules e.g. nitration of proteins (2) and the formation of nitrosothiols (3). Notwithstanding the array of reactions in which NO may participate and the wide range of proteins that can be involved it is arguably the case that the only physiological targets for NO itself are guanylate cyclase and cytochrome c oxidase. This proposition rests in part on the measured inhibition constants of NO for these two enzymes under the low oxygen tensions found in respiring cells and estimates of the NO concentration in vivo. These are in the 10 nM concentration range (see 4, 5, 6 for reviews). The fact that NO can bind to cytochrome oxidase has been known for almost 50 years (7) and given the avidity with which NO binds to haem groups it is unsurprising that it was subsequently shown that in vitro NO can bind tightly to ferrous heme a3 at the active site of the enzyme where oxygen Received 13 November 2003; accepted 1 December 2003 Address correspondence to: Michael T. Wilson, Department of Biological Sciences, University of Essex, Colchester CO4 3SQ, UK. Tel: + 44 1206 872538. Fax: + 44 1206 872592. E-mail:
[email protected] ISSN 1521-6543 print/ISSN 1521-6551 online # 2004 IUBMB DOI: 10.1080/152165430310001647293
can also bind (8). More surprising is the finding that NO reversibly inhibits cytochrome oxidase in cell culture and in vivo, competing effectively with oxygen. Strong evidence is now available showing that NO plays a true physiological role in vivo acting as an inhibitor of cytochrome oxidase (4, 5, 6, 9), a view supported by the recent report that mitochondria contain a functioning NO synthase (10). Given this context it is clearly of great importance to determine the mechanism through which NO inhibits cytochrome oxidase and how rapid release of inhibition may be achieved. The ways in which NO interact with cytochrome oxidase and the light that this throws on the inhibitory processes are briefly reviewed below. This discussion is also pertinent to the more general problem regarding the mechanism of reaction of NO with a wider class of copper based oxidases.
Cytochrome c oxidase Cytochrome c oxidase is the terminal electron acceptor of the mitochondrion and is responsible for the final step of the electron transfer chain in which electrons are passed to oxygen, reducing this to water. The reaction is complex, as full reduction of oxygen requires four electrons and these are supplied singly and sequentially by cytochrome c. Electrons pass from this donor to the acceptor site on cytochrome oxidase, CuA (a binuclear mixed valence copper site) and then to the coupled binuclear site comprising a high spin heme A group, termed here Fea3, and a copper atom, CuB, via a low spin haem A group, Fea. In order to achieve full reduction to water partially reduced oxygen intermediates are formed and these are tightly confined within the enzymes active site, bound to Fea3. Cytochrome oxidase rapidly and irreversibly reduces dioxygen by insuring that this ligand may only bind to the binuclear site when both the Fea3 and CuB sites are reduced i.e. Fea32 + , CuB + . In what amounts functionally to a concerted reaction three electrons are now passed to oxygen splitting the oxygen/oxygen bond to form one
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WILSON AND TORRES
CuB+ + NO22NO
H 2O
Pumped protons
e-
2e -
O NO 2
NO 2
R
NO
NO
NO
F
e-
P
oxyferryl
Fe2+a3NO
O2
H 2O
Figure 1. Schematic illustration of the sequence of turnover intermediates in cytochrome c oxidase. The catalytic cycle shows the intermediates at the binuclear Fea3CuB centre. The fully oxidized species O can accept 2 electrons to become reduced, R, and this species reacts with oxygen to form the oxyferryl species P and F. Electron addition returns the enzyme to species O. NO can react with a number of species. Reaction with O leads to reduction of CuB and electron transfer to FeaCuA. A similar reaction leads to reduction of P and F to yield O, forming nitrite. NO may also react with R. Not shown are the reactions of NO with the one-electron reduced binuclear centre.
molecule of water and leaving Fea3 in the oxyferryl state ([FeO]2 + ) and CuB oxidised to the cupric state (11, 12) (See Fig. 1). The Fea/CuA sites donate the third electron required for this but if these sites are oxidized then an electron is donated by a neighbouring aromatic residue forming a protein-based cation radical. Deprotonated forms of such radicals have been observed by EPR spectroscopy (13) and their location recently reanalysed and discussed (14). Addition of a further electron(s) from the Fea/CuA sites reduces this radical, if formed, and reduces the oxyferryl form to the ferric species, yielding the second water molecule. Accompanying the chemical reactions that form water the enzyme also couples the available redox energy to the transport of protons across the membrane, proton pumping. The mechanism of this pump and the steps in the catalytic cycle to which it is coupled remain the subjects of current research.
Reactions of cytochrome oxidase with NO In vitro studies of cytochrome c oxidase under turnover conditions confirmed that NO was competitive with oxygen and that inhibition was reversible (15). Similar studies on the purified enzyme also revealed, as perhaps expected, that the final form of the fully inhibited enzyme was one in which all sites were reduced and with NO bound to ferrous
Fea3, forming a nitrosyl adduct (16). However, the low apparent KI (*10 nM) of NO for cytochrome oxidase under these conditions, in which the electron flux through the enzyme was low and thus the enzyme in turnover largely populated states in which the binuclear centre was not reduced, lead to the suggestion that the effectiveness of NO as an inhibitor may rely on reaction with the binuclear centre when this was in states that do not themselves react readily or at all with oxygen. One possibility is that NO, unlike oxygen, reacts with the binuclear centre when this contains a single electron only. This hypothesis has found a measure of agreement although there is some debate over whether this electron resides on CuB or Fea3. Support for the latter view has been provided by extensive modelling of the mechanism (17), although the possibility that NO may also bind to the binuclear centre in which Fea3 is oxidised and CuB reduced remains live (16, 18). In addition to the hypothesis that NO reacts with the partially reduced binuclear centre is the possibility that NO may react with the enzyme’s turnover intermediates. This idea has now been tested by reacting NO with cytochrome oxidase when the enzyme resides in one or other of the available stable turnover intermediates (O, P or F see Fig. 1).
Reactions of NO with intermediates in the catalytic cycle The reaction of NO with the fully oxidized enzyme has previously been characterized as being very slow (19), however when the enzyme is freshly oxidized by oxygen (‘pulsed’ or O in the turnover cycle) its reactivity is very different. When freshly prepared species O, formed by oxidation of the fully reduced enzyme by oxygen, is mixed with a solution NO in a stopped-flow apparatus a rapid reaction ensues the rate of which is NO concentration dependent (20, 21). The second order combination rate constant has been determined as 2.2*105 M71s71 and pseudo first order rate of over 100 s71 are easily achieved at high NO concentrations (21). The most remarkable feature of the reaction however is the spectral transition that accompanies it (see Fig. 2). This unequivocally shows that the product of the reaction is the enzyme species in which Fea and CuA are partially reduced, sharing between them a single electron equivalent (to a first approximation) (22). In the presence of cyanide, which binds to ferric Fea3, the reaction is entirely suppressed. These results show that while NO binds to the binuclear centre the reaction is redox in nature leading to the partial reduction of the Fea/CuA centres. The simplest way to explain these findings is that NO reduces CuB and that the electron is then transferred from this metal to sites outside the binuclear centre. This being the case the reaction must lead to the oxidation of NO, probably through the transient formation of the NO + ion which on reaction with OH7 ions (or water) in the active site yields nitrous acid and hence nitrite. Nitrite has been identified as the product in
REACTIONS OF NITRIC OXIDE
Figure 2. Electron ejection from the binuclear centre on reaction of NO with species O. Cytochrome c oxidase, 4.7 mM, was reacted with 400 mM NO at pH 7. A rapid transition, k*100 s71, forming peaks at 605 and 445 nm allows this to be assigned to the reduction of Fea. NO reacts with Fea3CuB, reducing the copper that then transfers an electron to Fea. Rapid equilibration between Fea and CuA leads to partial reduction of the latter.
the active site (see Fig. 3) and is released into solution (22, 23, 24). Thus the oxidised enzyme (O) acts to metabolise NO to nitrite, equation (1). OH CuB 2þ þ NO ! CuB þ NOþ ! CuB þ þ NO2 þ Hþ
ð1Þ
This redox reactivity of NO is also exhibited in the reaction with the oxyferryl species F and P (20, 24). In these cases however the electron is not ejected from the binuclear centre but reduces the oxyferryl species to the ferric form yielding species O and nitrite. This means that NO may short circuit the turnover cycle by adding an electron to the binuclear centre, thereby bypassing steps involved with the proton pumping mechanism (see Fig. 1). In addition it has been shown that nitrite formed within the active site acts itself as an inhibitor of the enzyme (23). Thus NO has two modes through which it affects the activity of cytochrome oxidase, either by binding directly to ferrous Fea3 or by participating in a redox reaction with oxidized CuB forming nitrite. These two modes of inhibition have now been demonstrated to operate in Keilin Hartree particles and cell culture and are discussed further elsewhere (25). Inhibition by nitrite is itself complex because the release of this anion from the active site of the fully oxidized enzyme is very slow (t1/2 = 30 min), an observation in conflict with the known rapid reversal of NO inhibition. This problem is resolved by the finding that the rate of release of nitrite from the active site is enhanced if the Fea and CuA sites are reduced,
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Figure 3. Nitrite forms in the binuclear centre of cytochrome c oxidase on reaction of species F with NO. The broken line shows the difference spectrum between the product formed from reacting species F with NO and species O. The solid line shows the difference spectrum between species O that had been incubated with nitrite and species O. The spectra are essentially identical, indicating that the product of reacting F with NO is species O with nitrite within the binuclear centre. The oxidase concentration was 4.7 mM. The absorbance values in the visible region are multiplied by a factor of three compared with those of the Soret region.
i.e. as electrons enter the enzyme from the donor cytochrome c. This result may be rationalized by noting the requirement for electro neutrality of any transition that occurs in the binuclear centre, deeply buried within the membrane spanning subunit I of the enzyme (26). Thus for nitrite to diffuse from this centre requires either that it be replaced by an OH7 ion or be accompanied by a proton. The enzyme strictly controls the movement of these ions. If, however, the Fea/CuA sites are reduced there will be a driving force for an electron to enter the binuclear centre and this movement of a negative charge would expel the nitrite ion, relieving inhibition. This requirement for neutrality for transitions involving the Fea3/CuB sites probably also provides an explanation for the finding that although NO reduces CuB in the chloride bound enzyme (species O incubated with chloride) an electron does not leave the binuclear centre. To do so would require the chloride to be released to the bulk phase, which apparently is energetically or kinetically unfavourable. A similar situation may pertain in cytochrome bo3 in which it has been shown that NO reduces CuB but no electron is ejected from the active site, alternatively the relative redox potentials of the Feb site and the Feo3/CuB sites oppose the transfer. Evidence that nitrite does play a role in the inhibition of cytochrome oxidase by NO has also been derived from studies on the onset of inhibition (Torres and Wilson, unpublished data). In these experiments NO was added to cytochrome oxidase under aerobic conditions and in which electron donation to CuA was limited by a low concentration of
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WILSON AND TORRES
reduced electron donor, i.e. under slow turnover. In the absence of NO the enzyme populates the O, P and F states and the Fea and CuA sites are predominantly oxidized. On addition of NO the enzyme slowly became inhibited and it was possible to follow changes in the concentrations of the intermediates by monitoring their distinctive spectral contributions. It was similarly possible to follow the appearance of the final inhibited complex in which Fea3 is reduced and coordinated to NO, i.e. ferrous Fea3NO. Because electron entry was arranged to be slow it proved possible to follow the kinetics of these various processes. In particular rate of formation of the ferrous Fea3NO complex could be compared with the rate at which the enzyme became inhibited, judged by the rate at which Fea became reduced (indicating inhibition of electron transfer through the enzyme) and the rate at which nitrite was formed (indicated by the appearance of the characteristic spectrum in Fig. 4). The results of such experiments indicated that the onset of inhibition preceded the rate of ferrous Fea3NO formation, thus indicating the role of the redox reactivity of NO in inhibition of cytochrome oxidase at low electron flux.
Reaction of NO with laccase The reaction of NO with copper in at least one other copper containing oxidase indicates similar redox behaviour to that seen in cytochrome oxidase. Tree laccase contains four copper atoms traditionally classified as a single type 1 atom (T1, blue copper), a single type 2 atom (T2) and two type 3 atoms (T3) that comprise a magnetically coupled pair.
This enzyme has a broad specificity and can accept electrons from a range of donors. It uses dioxygen as the electron acceptor reducing this to water. Recent work suggests that oxygen binds and is reduced only when both the T3 pair and the T2 coppers are in the cuprous state, oxygen binding between the T2 copper and one of the T2 coppers (27, 28). Reactions of laccase with NO have been studied over a number of years and have suggested that these reactions are slow, taking some hours to complete (29). However, recently Torres et al. (30) have shown that in fact the T2 copper can react rapidly with NO in a redox reaction in which this copper atom becomes reduced. This reaction is difficult to monitor, being optically silent, and was only noticed because of particular optical properties of a partially reduced form of laccase. In this species the T3 coppers are reduced while T1 and T2 remain oxidized, and, according to Solomon et al. (see (28) for review), the enzyme is unable to bind oxygen. If this species is incubated anaerobically with NO and the product subsequently mixed with oxygen a rapid spectral change is observed. This transition is not observed if the partially reduced form is mixed with oxygen without prior incubation with NO. Analysis of the spectrum formed on adding oxygen, the kinetics of its formation and dependence on oxygen concentration allow the reaction to be assigned as the formation of the peroxide derivative of laccase. Given that this species can only form when the T2 copper is reduced Torres et al. (30) concluded that NO had reduced this copper making it available for oxygen binding. The overall reaction is illustrated in Fig. 5. We suggest that this type of reaction may not only hold implications for the regulation, by NO, of enzymes containing trinuclear copper centres (e.g. the major copper protein in blood plasma, ceruloplasmin), but also could be relevant to the regulation of copper enzymes in general.
CONCLUSIONS Although heme iron centres constitute the major sites at which NO reacts with metaloproteins, they are not unique in their ability to bind this important signal molecule. The
Figure 4. Inhibition of cytochrome oxidase by NO precedes formation of ferrous nitrosyl Fea3 at low electron flux. NO, 10 mM, was added to cytochrome c oxidase (4 mM) in the presence of reductant (ascorbate and ruthenium hexamine giving turnover no 0.2 s71) under aerobic conditions. Deconvolution of the spectra of the enzyme under turnover showed that the Fea3NO species (diamonds) was preceded by the appearance of the species in which nitrite is bound to species O (triangles) and the appearance of reduced Fea (squares).
Figure 5. Reactions of NO with partially reduced laccase. The active site of laccase comprises a binuclear pair, the type 3 coppers, and a type 2 copper. NO reduces the type 2 copper forming nitrite. Oxygen binds between cuprous T3 and T2 and electron donation to bound oxygen forms the peroxide intermediate.
REACTIONS OF NITRIC OXIDE
reaction of NO with cytochrome oxidase that leads to inhibition certainly involves coordination to ferrous Fea3 resulting in trapping this centre in the ferrous state and blocking electron transfer. However redox reactions can occur between NO and copper in copper-containing oxidases and these reactions should be kept in mind when considering the interactions of NO with such enzymes. When considering cytochrome oxidase it is probable that the reaction with CuB is physiologically important as it provides a way of metabolizing NO to non-toxic nitrite while still allowing rapidly reversible inhibition of the enzyme through two modes that operate at different electron fluxes through the enzyme. The mechanism is not fully understood, however, as the interplay between oxygen and NO concentrations and electron flux is still to be completely investigated. The redox reactions of NO at copper centres in such enzymes as laccase, that contain essentially a tri-nuclear copper that is found in enzymes such as ceruloplasmin suggest that such reactions may play a wider role in regulation and in NO metabolism.
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