Nitric Oxide-Some Old and New Perspectives Eric W. Ainscough and Andrew M. Brodie Department of Chemistry and Biochemistry, Massey University, Palmerston North, New Zealand Since its discovery and use by Priestley, nitric oxide has been cast a s both a villain and a hero of the chemical world. For example, in photochemical smog production and depletion of the ozone layer, it takes the former role (both are man induced, although NzO and NO do occur naturally); but in its conversion to nitric acid, fertilizers, and explosives, it takes the latter role. More recently it has been implicated a s a messenger molecule in neurobiology to help us retain long-term memory. I t also helps maintain blood pressure by dilating blood vessels, and i t helps to kill foreign invaders i n the immune response. Aspects of the chemistry or biology of nitric oxide related to these and other systems will be discussed. The Discovery and Use of Nitric Oxide Nitric oxide was among the first gases to be discovered. Priestley (Fig. 1) was credited with its discovery in 1772, but his work was based on experiments by Mayow as well a s Hayes (1). He obtained the gas, after asking Cavendish for advice, by the action of spirit af nitre (HN03) on a number of different metals, including iron, copper, tin, silver, mercury, bismuth, and nickel. The gas, called "nitrous air", was useful in his studies on the phenomena of combustion and respiration where he wished "to ascertain what change is made in the constitution of air by flame and to discover what provision there iiun nature ibr remedying the injury which the atmospherc n!wivrs by this means". He worked with confined volumes of air and rwnoved the "roodncss 01 air" (oxygen) by burning a candle, wax, or in it until the flame was extinguished or by mice breathing and eventually dying in it. With this foul air he performed various experiments and to monitor the state of the air after each experiment, he placed a mouse into it to see whether it would survive. The "goodness of air" was determined by whether the mouse survived. He showed t h a t a growing plant (in water) had the vower of restorine the air in which a candle had burned out'. Nitric uxiae forms deep red furnt:i of NO2 with air, and Priestlt:~used this chern~calresctlon to dt.t(!rmine the amount of of air". If NO is mixed with normal air over water, the NOz gas is absorbed by the water with a diminution of volume amounting to about one fifth of the air. The diminution in volume was in proportion to the amount of oxygen in the air. The "goodness of air" hence could be distinguished more accurately than the biological testing with mice which Priestley disliked.
woos
-
Flgure 1 Josepn mestley 1772 and oxygen in 1774
(1 133-1804)
discovered nltrlc oxide in
Preparation of Nitric Oxide (2) Nitric oxide is a colorless gas formed in reactions involving reduction of nitric acid and solutions of nitrates and nitrites. For example copper metal is used a s a reducing agent in 8 M nitric acid according to the equation:
ii
The commercial route to NO is by means of catalytic oxidation of M I 3 (Fig. 2) which is the first step in a three-step process known a s the Ostwald process by which NH3 is converted into nitric acid.
Figure 2. The platnum-rhodium gauze catalyst used in the oxdation of ammonia to nitric oxide. one oithe steps in the production of nitric acid.
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Journal of Chemical Education
The first very efficient step is according to the following reaction: Pt - RhCatalvst
when weather conditions produce a relative stagnant air mass. Catalytic mufflers are designed to reduce the levels of NO, ix = 1 or 2) and hydrocarbons. For the former, a catalyst based on noble metals has been produced to affect the reactions:
On exposure to air the cooled gases react to produce the red-brown gas NOz, according to the equation:
When dissolved i n water, NOz forms nitric acid in a disproportionation reaction:
The NO can be converted back to NOz to prepare more HN03. The first plant was set u p in Germany in 1908, and Ostwald was awarded the Nobel Prize in 1909. This allowed the large-scale synthesis of KN03 a n d NH4N03 which are used a s explosives and fertilizers. In earlier times KN03 was found i n pigsties or manure trenches where it was produced by the action of bacteria in dung and soils with lime and urine. The first step in the Birkeland-Eyde process (1903) for production of HN03 involved sparking nitrogen and oxygen according to the equation: Spark
Nz(g) + Oz(g) -->
2NO(g)
However, the yield was low and the process expensive and consequently is now obsolete. Nitric Oxide and Photochemical Smog The above Birkeland-Eyde reaction i s a significant source of nitrogen oxide air pollutants which form during combustion reactions in air. NO is formed in automobile internal combustion engines and then converted to NOz in air. In sunlight, NOz undergoes dissociation to NO and 0 : 393 nm
NO&) + hv
> NO(@+
I n this simplified sequence atomic oxygen formed nndergoes several possible reactions one of which forms ozone:
The activity of catalytic converters decreases with use due to poisoning etc., and a really efficient catalyst has still to be found. Recently, a Cu2+exchanged ZSM-5 catalyst has shown high and stable catalytic activity for NO decomposition to Nz and OZ.(ZSM-5 is a high silica-containing aluminosilicate with a framework enclosing cavities occupied by cations and water molecules. I n this case Cu2+ions OCCUPY some of the cavities). Nitric Oxide and Ozone Depletion Problems also occur when NO is present i n the stratosphere, the region between 10 and 50 k m from the Earth's surface. Ozone reacts with NO to form NO2 and Oz; then NOz reacts with atomic oxygen to regenerate NO and Oz according to the equations:
O&) + NO(& 4 NO&) + Oz(g) NOz(g) + O(g)+ NOW + Oz(g1 0 3 + OW + 20z(g)
NO is a catalyst which serves the function of increasing the rate of decomposition of 0 3 which is used to filter off solar radiation of less than 300 nm wavelength. Ozone is used to protect Earth from the harmful effects of such radiation, and life on Earth would not he possible without it. Concern has been expressed that as supersonic aircraft fly in the stratosphere and emit NO in their exhaust fumes, this also could reduce the ozone concentration. Chemistrv in thv stratosphere IScompit,x and not well undrrstood. The reaction d o z o n e w i t h KO is :I chtmiluminesccnr reaction, and in the laboratory can be used to determine NO concentration. Some NOz molecules are produced in an excited state, and when these decay to a ground state the luminescence produced is proportional to the concentration of NOz in the excited state. Selected Physical and Chemical Properties of Nitric Oxide (3 Nitric oxide i s a neutral oxide with the electron configuration
( 0 2 ~(028)~ ) ~ ( 0 2 ~( ~~) ~ 2( ~~2 6) )~~
(M= any third body e.g. Nz or 02) Ozone is a powerful oxidizing agent, is very irritating to the eyes, causes damage to the respiratory system, and its presence in the troposphere is harmful to plant life. (The Earth's atmosphere may be divided into a number of regions, the lowest layer called the "troposphere" extends from the Earth's surface to a n altitude of about 10 km.) I t can convert NO back to NOz. O&) + NOW + NO,(g) + O&) NO in the troposphere is also converted to NOz by reaction of hydrocarbon peroxy radicals (ROz). Along with olefins, aldehydes (produced by reaction of the olefins with NOz or 031, C2H2,CO, CH4, peroxyacylnitrates R-C-0-0-NOZ
The unpaired x* electron renders the molecule paramagnetic with a bond order of 2.5.I n the solid state NO dimerizes to O=N-N=O which is diamagnetic. NO is moderately to reactive and reacts with Clz to produce NOCl and 0%produce peroxonitrite (-OONO) a t pH 12-13 which a t low pH decays to form HN03. If the electron in the (~2;)
orbital is removed by oxidation, the nitrosoninm ion NOt is formed with the N-0 bond order now three (isoelectronic with nitrogen). The N-0 vibration frequency rises from 1840 cm-' i n NO up to 2300 cm-' for NO'. Priestley first converted nitric oxide (oxidation state o f N = 2) to nitrous oxide (oxidation state of N = 1) by reduction with moist iron filings according to the equation:
II
6 etc. the mixture is described as photochemical smog which pollutes the air in places such a s Los Angeles, particularly
though i t i s not the most convenient synthesis. NzO is a relatively unreactive gas and was used by Davey a s the Volume 72
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first anesthetic, and it is used a s a n aerosol propellant because of its solubility in lipids such as cream. Similarly, NO can diffuse easily across cell membranes. The union of equimolar quantities of NO and NO2 a t low temperature gives the blue unstable liquid Nz03 (oxidation state of N = 3):
of nitrite, and i t has been found in cytotoxic mouse macrophages a s well (8,Y). Another use of nitric oxide is in its ability to give meat a fresh red appearance. I n the absence of nitrite (or nitrate) meat darkens in color because the iron in myoglobin is oxidized. NO, produced from reduction of nitrites (or nitrates) combines with myoglobin (Mb) to give a red-colored compound which retards oxidation:
When an equimolar mixture of NO and NOz is passed into cold water, nitrous acid (HN02)is formed: N 2 0 ~is the anhydride of HN02. Even in cold solutions some disproportionation of HNOz to HNO,, NO and water occurs. Nitrites, the salts of nitrous acid, can be made by passing a n equimolar mixture of NO and NO2 into a metal hydroxide, according to the equation:
Nitrites a s Food and Other Additives Nitrites (and nitrates which are converted to nitrites) are used a s preservatives and curing agents in meats such a s bacon, sausages, hot dogs, and tinned ham (3, 4). Though approved, their use is controversial. For NaNOz the tolerance limit for humans is 5-10 g per day depending on the body weight. A concern is that ingestion of nitrites in the stomach leads to formation of the [HzONOlt ion which is a powerful nitrosation agent. This results in the formation of nitrosamines that can lead to cancer. Nitrite ions inhibit the growth of bacteria-particularly Clostridium botulinum-which causes botulism, a serious form of food poisoning. The bactericidal mechanism of nitrite inhibition may involve the inactivation of iron-sulfur enzymes, especially the electron-transfer protein ferredoxin (5).Vegetative cells of Clostridium botulinum were treated with sodium nitrite and ascorbate and studied by electron-svin resonance (ESR). Untreated samoles exhibited an 1.:h signal rharactcristic of it high poientaal iron protein with an iron-sull'ur center IS = 1.9 13 which rhnnaed In the treated sample to one characteristic for nitroUsyl (NO) complexes of iron (g = 2.035) (6). Ascorbate ions reduce nitrite ions to NO which react with the [ F ~ ~ s ~ ( s R ) & cores of the iron-sulfur protein to produce possibly the [Fe(SR)z(NOjz]- ion which displays the new ESR signal (6). The first role of NO is to destroy the appropriate enzymes, and the second role is to inhibit microbial growth. However, a more recent view is that NO'S antibacterial properties also may arise a s a result of the formation of NOz and OH radicals which result from decomposition of the product formed by reaction of NO with the superoxide ion Oz- and Ht. Both are killers of cells. Not all reactions of nitrite with iron-sulfur proteins are beneficial. The incidence of esophageal cancer in Chinese citizens living in Linzian has been attributed to the consumption of cornbread and vegetables pickled by storage in water for several weeks. The water contained elevated levels of n i t r i t e a n d n i t r a t e ions. The diamagnetic, Roussin's red ester [Fez(SMe)z(N0)41, has been isolated from the vegetables (7) and, although it is non-carcinogenic, i t can potentiate the action of other carcinogens. The cornbread often is contaminated with a mould which, in the presence of nitrite, can produce a range of N-nitrosamines. The two distinct types of nitroso compounds together may be deleterious to health. A similar ESR signal to the [Fe(SMeMN0)21- ion has been reported to be associated with tumor formation in animals induced by administration of chemical carcinogens and enhanced by the addition
688
Journal of Chemical Education
Nitrite ions alsc~m.iv oxidize the Ft!'--porphyrin in Mh and hcmoglohin Hb, thus ftmning tli' red-nttrozo porphyrin derivauvc t Ni) b',,"Mbor .NO!E'(.'"Hb. In ~~~-~ h -I.c',,- 111,state . ...... these porpbyrins have no oxygen-carrying ability and a s well NO occupies the sites. I n babies or infants the consumption of N W N 0 5 -containing foods can create a n oxygen-deficiency crisis with the name methemoglobinemia. One of the concerns of the use of nitrites is that during the cook in^ of bacon or the smokinn .. of fish thev mav react with nmines and be c o n v r r t d to nitrosamint!~ R2-SS = O IIns shown below. u,hirh i r e ~houghtto induce c;inwr. Some form of preservation of food is necessary, but i t is not clear bow unsafe the present reagents are. Finally, it is known that canned fruits may contain elevated levels of iron due to solubilization of the metal from the containers. The pH and the presence of nitrate in the canned food are important determinants of the amount of iron dissolved. The iron reduces the nitrate ion to NONO2 (see preparation section). Some fruits such a s papaya can have such high levels of nitrate that they cannot be canned without excessive iron solution. Nitrosyl Coordination Complexes (2) Nitrosonium [ N u ] Complexes As shown above NO readily forms coordination complexes with transition metal ions, and these are called nitrosyls. An iron nitrosyl complex, the ruby-red sodium nitroprusside, Naz[Fe(CN)sNOlC2HzO r e s u l t s from t h e action of nitrite ion on the [ F ~ ( c N ) ~ Iion. "
The complex anion is diamagnetic and contains a n N-0 stretching vibration a t 1939 em-', higher than for NO itself. The anion contains a linear FeNO group and has been formulated a s a NOt complex of Fe(I1). In a linear MNO structure the basic interaction is by donation of a o pair of electrons from N to iron with a covalent Nn*-dn bond with some additional bonding due to dn +Nn* interaction. NO is a three-electron donor in this situation. A net positive charge on the NO raises the NO-stretching frequency above that for the free NO molecule. The metal-nitrosyl complexes above can be viewed a s NOt carriers. Other examples are thionitrites (RS--NO) obtained by reaction of NOt with thiolates RS-, and nitrosamines (RzNNO) formed a s described above. 1'n biology tbionitrites have been detected. and thev are viewed as a means to 'package2N0in a fo& better suited to its intermediary roles: ex., - .vrolona life in the blood and tissues and fire-ililiitt. trnnsport. ~i;ccr).l nitrate ,see latcr, rnetabol~sminvdvt!.+ format:on of S-uitro.+~thiols.which rnav serve as precursors to NO. Nucleovhiles such a s OH-. RS-. and S~ ions attack ~~-the ~--positively charged N atom of NO; in the nitroprusside ion to give the following products: ~
~~~
The nitroprusside ion reacts with the sulfide ion to give the purple anion, possibly [ ( c N ) ~ F ~ ( N o s ) I while ~with the RS-, the red
ion is formed. Both reactions form the basis of sensitive tests for the detection of the S2-and RS- ions. Nitroxyl Anion (NO3 Complexes These have received less attention, hut in the complex [C~(NH~)~(NO ion ) ] the ~ + CoNO group is bent (CoNO angle 119') and the cobalt is treated as Co(II1) and the NO a s NO-. The N-0 stretching vibration occurs a t 1610 cm-' which is low. In this situation the N O species is a one-electron donor, and the N-0 bond order i s decreased (the added electron occupies the ,726
orbital); and the N-0 frequency is decreased. Uncomplexed NO-spontaneously dimerizes to form N20 and Hz0 in the presence of protons. In the 'brown-ring test' for nitrate, NO is produced by reduction of the nitrate ion by ~ e "in acid solution according to the equation: NO,+ 4H' + 3e -t NO(g)+ 2H20 The NO reacts with more ferrous ions to produce a darkcolored compound according to the reaction:
This compound is unstable and hence imposes difficulties on eventual X-ray analysis. However, the crystal structure of [Cr(H~0)5NOlS04i s compatible with a chromium(II1) complex of NO-, and the iron complex also is thought to contain NO- species. The N-0 frequencies for these two complexes lie i n the range 1733-1765 cm-' which is indeterminant for discrimination hetween "NO'" and "NO-". Sodium Nitroprusside and Other Nitrovasodilators Sodium nitroprusside (Nipruss or Nipride) is used a s a relaxant of the muscles in the walls of the arteries. This results in a widening of the arteries and a reduction in blood flow. In the treatment of cranial aneurysms (a condition where the outer layers of the wall of the blood vessel have weakened or been lost and the inner layer swells, and if the swelling bursts, a hemorrhage could result) a dilute solution of this drug i s passed into the patient's blood stream, a n d there i s rapid vascular muscle relaxation (1Oa). The defective length of blood vessel is removed during t h e surgery. The drug is also used in heart transplant clinics.
If the heart's own blood flow is restricted because of narrowing of the arteries then this condition places more strain on it, and a lack of blood and oxygen reduces the ability of the heart to act as a pump and results in the victim becoming breathless and being i n great pain-a condition called angina. Amy1 nitrite (lCH3)zCH-CHz-CHz-ON01 and glyceryl nitrate ( ~ z N O - C H ~ ~ H ( O N ~ Z ) - C H ~ Oa rNe O Z ) used for quick treatment. The use of the former was discovered in 1867 by Brunton while the use of the latter was noted after girls who packed explosives during World War I had abnormally low hlood pressure (IOU).These compounds (called nitrovasodilators) result in an enlargement of the blood vessel by smooth muscle relaxation, reducing blood pressure. It is generally accepted that many nitrovasodilators mediate muscle relaxation by producing NO (which would have to he derived for a n NOt-containing vasodilator) which activates guanylate cyclase. This enzyme contains a n iron-heme component that is vital to the mechanism. The binding of NO to the heme triggers the release of the proximal histidine ligand allowing the free imidazole to act as a general base catalyst or the breaking of the iron-imidazole hand triggers a conformational change of the enzyme. Either way the enzyme is activated to increase guanosine 3',5'-monophosphate production t h a t enhances transmitter (glutamate) release, which affects vasodilation (as well a s neurotransmission, digestion i n the gut and penile erection). Nipruss also has been used a s a n emergency drug in cases of high hlood pressure crises. Nitrovasodilators are not the onlv com~oundsthat effect arterial muscle relaxation. ~ c e ~ ~ l c h ~(CH3CO-Oline CHZ-CHZ-N'(CH~)~)a n d bradykiuin ( a nonapeptide) have a similar effect. The former has a reducingeffect on rabbit thoracic aorta (meat arterv issuine, from the heart). Cells on the inner &ace of the Gessel incontact with the hlood a s i t is pumped along the aorta are called endothelial cells, and these may release an endothelial-derived relaxing factor (the EDRF). This i s true for acetylcholine, but if the cells are removed the effect is destroyed (10). The acetylcholine stimulates the endothelial cells to produce 'messenger' species, and these activate guanylate cyclase to bring about muscle relaxation. The 'messenger' species or the EDRF by analogy with many nitrovasodilators is now thought to be simply NO. Initially, this suggestion was startling, but experimental evidence for the production of NO is available. One experiment involved a culture of arginine-deprived endothelial cells being fed with L-arginine-containing ' 5 a~t one of the terminal positions. The culture was stimulated to produce 1 5 ~by 0 bradykinin (lob). NO thus appears to he a natural messenger for control of blood flow by vascular muscle relaxation. In solution and a t very low concentrations the reaction with oxygen must be quite slow, otherwise the above-stated biological role would he unlikely Nitric Oxide in Other Biological Systems DNA Deamination Some human genetic diseases and cancer are thought to arise from deamination of DNA, yet hydrolytic deamination i s unfavorable energetically with cytosine residues having a lifetime of 30,000 years in the duplex. Certain agents may catalyze this reaction. One agent i s NO, an airpollutant, a cigarette smoke constituent, and a recently discovered bio-regulatory agent (see above) produced in many cells. I n uitro NO has been shown to deaminate the bases in deoxyuucleosides, deoxynucleotides, and intact DNA. Similar DNA damage also can occur in vivo. Reactions typical of the chemistry involved are given (11).
Volume 72 Number 8 August 1995
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(Ar = aromatic)
Ar$
+ H,O
-->
ArOH + N,
+ H+
This reaction would result in the base cytosine being converted to uracil and methylated cytosine to thymine. These and other transformations would give nonreparable lesions.
~re-synoptic neuron
0 0
Long-Term Memory Astudent studying for an examination Figure 3. In the retrograde hypothesis for memory storage, nitric oxide made by the post-systores information away i n long-term naptic neuron may feed back to the pre-synapticneuron telling it to increase its output of neuromemory. Somehow neural connections transmitters. corresponding to new knowledge are being strengthened. How this occurs is not NO synthase like cytochrome P450, contains iron-proknown with certainty, but a process called long-term potoporphyrin IX. A five-electron oxidation of a terminal tentiation (LTP) is being investigated (12, 13).Alterations guanidino nitrogen of arginine is supported by reduced niunderlying long-term memories take place a t the syncotinamide adenine dinucleotide phosphate (NADPH) and apses, the points where signals pass from one nerve cell to other co-factors. One oxygen molecule hinds to the Fe(I1) the next. The question t h a t researchers are asking is site and after transfer of an electron from NADPH to the whether the chances take place in the cell that sends the heme, scission of the 0-0 bond occurs, with one atom of sihmnl or in the rell arross the sj napse that rrcuivc,s it. One oxygen inserting into one of the terminal N-H bonds of arrrctm model (12, 13 oroooses that the nerve cell on the ginine. After reduction of the iron to Fe(I1) a second molereceiving end of a message would send a 'retrograde mescule of oxygen binds and this species may act a s a n oxidant senger'back to the sending cell, strengthening the connecremoving a n electron from the N-hydroxy-arginine to gention between them and contributing to the formation of a erate a peroxo-iron species and a N-hvdroxv-arpinine catlong-term memory (Fig. 3). There appears to be some eviion radical. Attack by the peroxo-iron speciis on-the cation dence that nitric oxide is a key chemical player in the storradical a t the gnanidino carbon finally yields L-citmlline age of memories in the brain; i.e., it may be the "retrograde and NO. The remaining oxygen atomaappear a s water. messenger" (12,131. If this is the case it would be one of the more bizarre molecules used bv cells. I t does have the ahility toslip right thrungh cell membranes, and it does disapISH+ near within moments of its ~roduction.Thc NO m;i\. .ict on Ei'H2 7-02 b a n y l a t e cyclase or ADP Ab~s~ltransferase in the presyy 2 C=NH I .5 NADPH uaptic cells. i HPC-(CH~)~-NH LTP is triggered when a neuron receives several simuli NO synthase taneous signals. The signals trigger a class of L-glutamate COOH receptors to let calcium into the cell. The calcium causes the synapses that delivered the signals to be strengthened. L - arginine This leads to "potentiationn- a bigger response in the receiving cell the next time signals are sent. Nitric oxide is suggested a s a messenger from the postsynaptic (receivEi'H2 ing) cell back to the presynaptic (sending) cell, telling the yH2 C=O sender to increase its output (Fig. 3) (13). i The nitric oxide-producing enzyme NO synthase is inH-C-(CH33-NH +NO + 2H,O + 1.5 NADP+ i hibited by infusing hrain slices with hemoclohin. The heCOOH moglobil;does not.pc:nt,tret(r wll membranes hut binds the NO c~xtr:ic(.llularl\:Sodium nitroorusside, a compound L - citrulline that can release i t i NO, enhances synaptic transmission. Studies using 1 4 and ~ '5~-labelledterminal guanidino The detection of 1 4 ~may 0 be done by chemilnminesnitrogen atom(s1 of L-arginine clearly demonstrate that Lcence (after reaction with ozone) by reaction with O r arginine is a substrate for the generation of NO by the acwhich forms NO3f or a s 1 5 ~by 0 mass spectrometry. Retion of NO synthase (14-17). The precise mechanism cently a n NO sensor has been developed which relies on whereby NO is formed is not known. One suggestion is the interaction of NO with polymeric porphyrins containthat deimination leads to formation of NH3 which is oxiing nickel (30). CO may also function in a similar way to dized to NO. L-citrnlline is also ~roduced(9. 18-20). NO in LTP More importantly, a second su&estion is that the termiNO is essential for the complex mechanism involving nal gnanidino nitrogen atom(s) of L-ar!zinine mav be N-oxinerves, muscles, and blood pressure required for a male's dizeb in a reactioninvolving enzyme-mediated attack by sexual potency. I t also relaxes the muscles of the intestine oxidizing s ~ e c i e such s as oxvpen radicals or l i ~ i oeroxides d so food can be shunted along by muscular contraction and (15). ( T G ~N O synthase fo&d in activated macrophages relaxation (9, 11, 15,311. has different ~ e s that found in neurons in the . r o. ~ e r t ifrom Anitric oxide synthase has been found in the blood cells bram. The former functions indvp(!ndmtly d'ml(:inm and of horseshoe crabs and the blood sucking bug, Rhodnius calmudulin which ;!re reanired hv the latter., Calmodul~n prolixus, has a vasodilator in the salivary gland, which is binds to nitric oxide synthase in brain neurons after a n colored bright red owing to the presence of an Fe(II1) heme increase in intracellular calcium. This activates NO production (21). that binds NO. Dilution of the protein in neutral pH pro-
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Journal of Chemical Education
moted NO release, which helps the bug to feed on blood (32). NO is also synthesized by fruit flies, chickens, barnacles, and trout. NO in Neuronal Destruction and Protection in Vascular Stroke and Neurodeqenerative Diseases NO may also mediate major neuronal damage in stroke and neurodegenerative diseases. Neural destruction in stroke seems to result from a massive release of glutamate which a d ing through the N-methyl-D-aspartate (NMDA)subtype of receptor somehow causes "excess excitation" resulting in neuronal death. NO which mediates the neurotoxic effects of glutamate is formed in large amounts and results in neuronal death. Lipton et al. (33)suggest that the neurotoxic actions of NO derive from its readion with the supemxide anion to form peroxonitrite, which may be the neurotoxic agent. I n some studies, however, NO seems to be neuroprotective. Lipton e t al. (33)suggest t h a t NO in the form of the nitrosonium ion (NOt) reacts with the thiol group of the NMDA receptor to block neurotransmission. A great effort to develop NO synthase inhibitors a s antistroke drugs is underway. Similar considerations would apply to drugs aimed a t dementia and neurodegenerative conditions such a s Huntington's and Parkinson's diseases which may also involve excessive stimulation of NMDA receptors.
Immune Response White blood cells known a s macrophages make NO to defend the body from microorganisms and to attack cancer cells. This occurs, e.g., when they receive signals such as liposaccharide from bacteria or gamma interferon from immune cells. NO'S antibacterial properties may be due to the formation of the cellular killers, the hydroxyl radical, and NOz which arise after decomposition of the product formed by reaction of NO with the superoxide ion and Hi. NO'S antitumor function may involve the inhibition of metabolic pathways to block growth. NO attacks iron groups in certain enzymes, including those that synthesize DNA and when these enzymes are crippled the cells cannot grow. Bacterial Denitrification of Nitrates and Nitrites The interactions of nitrogen oxides with copper containing enzymes in biological systems are important in the global nitrogen cycle. I n these systems bacteria (in soils) use NOj and N@ ions a s terminal electron acceptors to produce NO, NzO, and Nz (22a). I n nitrite reductase from Achromobacter cycloclastes the NO2 ion is believed to bind a t a single copper (11)ion that is coordinated by three histidines and a n aquo or hydroxo moiety, in a most unusual pseudotetrahedral array (23). I t is inferred that the N@ ion enters a 12 A deep solvent channel, displaces the aquomoiety, binds to the copper(1) and in the presence of protons produces E-Cut-NOf (E = enzyme) according to the reaction:
(24,251. I t is also in equilibrium with E-CU'+ and NO. The next stage may be the unusual interaction of E-CuWOt with N@ ions and electrons and protons to produce NzO as shown:
An equally possible and a t present a non-distinguishable alternative scheme involves the reaction of E-Cut-NOt with NO (assuming its original release is rapid) along with protons and electrons according to the equation:
The nitrite reduction to NO and NzO occurs a t the same copper site which is classified as a putative type I1 site. Another copper, which is located about 12.5 A away, is classified as a type I site and has a flattened tetrahedral geometry. This provides the type I1 site with electrons for the reduction. For the type I1 copper site to receive its full complement of histidine ligands the enzyme exists as a trimer (one histidine comes from each monomeric unit). A copper(1) p s e u d o - t e t r a h e d r a l compound w i t h stoichiometry N3CuNO (N3 is a n anionic triad of heterocyclic nitrogen donor ligands) has proved a suitable model for the reduced type I1 active site (26). This is the first nitrosyl complex of copper and has a Cu-N-0 angle of 163.4' and a u(N-0) stretch a t 1712 cm-'. The Cu-N(0) distance is short indicating the presence of some degree of multiple bonding. The ability of this NO complex to undergo reaction to yield NO and NzO has been demonstrated. A nitrous oxide reductase from Pseudomonas stutzeri has two identical subunits, each subunit canyingfour copper atoms (27). The reductase is the terminal electron acceptor in a respiratory chain converting NzO to Nz: N20 + 2H+ + 2e 3 N2 + H20
Binuclear, antiferromagnetically-coupled copper(I1) sites are involved. Other nitrite (and nitrate) reductases are proteins containing iron chlorin or isobacteriochlorin; however, less is known about these (226) and their mode of action is different from the copper enzymes. Epilogue Two hundred and twenty years after Priestley discovered and used NO in his studies on combustion and photosynthesis, there has been a re-birth of interest in NO (and its redox activated forms) because of its unexpected roles in physiology and neurobiology, and how these are dependent on NO not reacting with oxygen. NO is produced from the reaction of NO synthase with L-arginine, and i t activates guanylate cyclase to produce guanosine 3', %monovhosvhate which can lead to biolopical resvonses such a s vasodilation, cell adhesion, neurotransmission, and penile erection. Bv other interactions it can affect enzvme and immune r e G a t i o n and cytotoxicity. Further probing of NO'S activities in the brain, arteries, immune system, liver, pan-
. .
The copper-NO species (E-Cuf-NOt) is suggested to be a key intermediate in biological nitrogen oxide reduction
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creas, periphereal nerves, and lungs will ensure that there will remain much interest in the old gas in the future. NO may represent a new class of signalling molecules; i.e., gases that swiftly pass through cells and vanish. Under physiological conditions NO can be interconverted among different redox forms with different chemistries. The myriad of activities attributed to " N O may possibly also be attributed to other forms, but this is not a consensus opinion (2830). Overactive neurons produce large amounts of NO and this may be linked to certain brain diseases. Also a condition called septic shock occurs when some bacterial infections cause the endothelium to release large amounts of NO. The patients'blood vessels dilate and a life-threatening lowering of the blood pressure occurs. Microbes convert nitrates or nitrites to NO, N20, and Nz, but NO emission from the internal combustion engine has been controversial because of its involvement in the generation of photochemical smog and ozone depletion. Literature Cited 1. Partington, J . R. AHistoq of Chemistry; Memillan Press: London, 1962. 237. 2. Cotton, F.A.: Wilkinson, GAduonndInorgonicCh~mistry,3rded.; Interscience Publishem: New York. 1972. 3. Pimick, H.; Johnston, M. A ; Thacker C.; L a p s R. Con. Inst F m d Sci. nchnol. J 1970.3, 103. 4. Johnston. M. A : Plunick, H.; Samson, J . M. Con. Insf. Food Sci. Tkhnoi. J 1969.2, 6"
5. Reddy D.: h a a t o r , J. 8.; Cornforth, D. P. S c h n 1983,221,769. 6. But1er.A. R.: Glidewell, C.: Hyde,A.R.:Wdton, J. C.Polyhadmn 1985,4,797. 7. Wang, G. H.; Zhang, W X.; Chai, W G.Aclo Chim. Sinlco I980,38,95; Glidewell, C. Chem. Br 1990.26.137.
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, E. 8. Emanuel, N. W.: Saptin, A. N.: Shsbalkin, V. A.; Kozlova. L. E.; K ~ g l j a k o v s K. Nolure 1969,222, 165. 9. Lsncasfer, J. R.Jr.: Hibbs, J. B. H. Jr ProeNotl. Acod. Sci. USA 1990.87.1223. 10. (a1Butler.A. R. Chem. Br. 1990.26.419, ibl P d m e s R. M. J.;k.hton, D. C.; Moneada. S. Nature 1968,333,664. 11. Wink,D. A.;Kasprzak, K S.:Marago$. C.M.: Elespuru,R. K;Misra,M.; Dunan8.T. M.: Cebu1a.T.A: Kach, W. H.;Andrews.A. W.;Ailen. J . S.:Keefer,L. KScience 1990,254,1001. 12. Schuman. E. M.; Madison. 0.Y Science 1991.254. 1503. 13. O'Dell, T. J.:Hawkins, R. D.; Kmdel, E. R.;Arancio, 0. Pmc Noli.Amd. Sci. USA 1991,188,11286. 14. Palmer, R. M. J.: Fertige, A. G.; Moncsda, S. Nature 1987,327,524. 15. Palmer. R. M. J . ; h h t o n , D. S.; Moneads,S.Nature 1388,333,664. 16. Bredt, D. S.; Hwang, P M.; Snyder, S. H. Nolum 1990,347,768, 17. Darlhwaite. J.: Charles, S. L.:Chers-Williams, R. Nafun 1988,336, 385. 18. lyengarR.;Stuehr,D. J.;Marletta,M.A.Proe. Nofi.Arnd. Sci. USA1987.84,6369. Is. Moncsda. S.: Higgs, E. A. Nitric Oxide from L-Argmine: A Biore#~clatooSystem; ElsevierlExerpta Medica: Amsterdam, 1990. 20. Hibbs. J. B.; Taintor, R. R.; V h n , 2.Science 1987,235,473, 21. Xie,Q.:Heam, J. C.;Calaycay J.;MumfodR.A.:Swiderek. K. M.;Lee.T. D.:Dlng, A.; l h s o , T.: Nathm, C. Seienen 1992,256,225, 22. iar Denirnlicoaon. nirn/icotion, ond~rmospherienitmus OT~&;~ d w h i c h e C. , c., Ed.: John W h y and Sons: New York, 1981. tbl F e w s o n , S. J. l k n d s Bioehem. Sci. 1987, 12, 354. J.:LsGall, 23. Godden. J.W.:~riey,S.:Teller,D.C.;Adman,E.T.;Liu,M.-Y;Payne,W J . Science 1991.253.438. 24. Jackson. M. A ; Tiedie; J. M.; Avellll, B. A FEES Loit. 1991.291,41. J. M.;Avedl,B.A.JBioi. Chem. 1991,266, 12848. 25. Ye.8. W;To&uarel,I.:Tiedje, 26. Cartier, S. M.; Rugeem, C. E.: Tolman, W B.: Jameson. G. B. J Am. Chem. Soe 1992. I I C 4401. 27. Coyle. C.L.:ZumR, W. 0.:Kroneek. P M. H.; Kormer,H.; Jacob. W E u r J.Biaehem. 1985., 157.419~ ~ ~ ~ , ~ ~ 28. Culotta, E.; Koshland, D. E. Sezence 1992,258. 1862. 29. Stamler. J. S.: Singel, D. J.; Loscalzo, J. Seienm 1992.258. 1898. 30. Malinski. T: Tads, 2. Nofun 1992,358,676. 31. Young, S. New Scmntid 1993,137 tNo 18641.36, 32. R~beira,J.M. C.; Hszzsrd, J. M. H.:Nussenzueig,R.H.; Champagne. D. E.: Walker, FA. Sclcnce 1993,280,539. J.:Loseaho. 33. Liptan.S.A.;Choi.V.B.:Psn.Z.H.:Lei,S.Z.;Chen.H.S.Y;Sucher.N. J.: Singel, D. J.: Stamler, J. S. Nolure 1993,364,626,
