SPECIAL FOCUS y Nitric oxide-releasing materials

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1 Special Report 2015/06/30 Nitric oxide: a brief overview of chemical and physical properties relevant to therapeutic applications

Future Sci. OA Nitric oxide (nitrogen monoxide, •NO) has been intensively studied by chemists and Jack R Lancaster Jr physicists for over 200 years and thus there is an extensive database of information Departments of Pharmacology & that determines its biological actions. This is a very brief overview of the chemical and Chemical Biology, Medicine, & Surgery, School of Medicine, University of physical properties of •NO that are most relevant to Biology in general and to the Pittsburgh, 4200 Fifth Ave, Pittsburgh, 1(1), FSO59 development of •NO releasing materials in particular. PA 15260, USA [email protected] Keywords: diffusion • oxidation-reduction • radical • transition metals

In 1987 the startling discovery was made electron. This is due to the fact that it has that the small reactive radical molecule an odd total number of electrons (15). The nitric oxide (•NO; nitrogen monoxide) is property of possessing an unpaired electron specifically produced and functions as a is called paramagnetism and it is this prop­ major messenger in the mammalian cardio­ erty that most determines its chemical reac­ vascular system, for which the 1998 Nobel tivity. However, the small uncharged nature Prize in Physiology or Medicine was awarded of •NO is especially important in determin­ to Robert Furchgott, Louis Ignarro and Ferid ing mechanisms for its selective delivery, the Murad [1] . This discovery represented a classi­ topic of this monograph. cal paradigm shift in chemical biology, reveal­ This brief overview is organized into two ing the profound concept that Nature utilizes general topics, the chemical and the physical to its advantage the properties of molecules, properties of •NO relevant to its biological even if they can also be injurious or toxic. actions. Prior to this realization, •NO was known to biologists almost exclusively as only a Chemical properties of •NO: a lone toxic pollutant and we now know it is truly electron in search of a stable home 2 a versatile player in virtually every aspect of Unpaired electrons in σ or π molecular ­physiology and ­pathophysiology. orbitals are inherently unstable. The two The proposition that the fundamental methods for the stabilization of this elec­ principles of biology must ultimately be tron are pair with another unpaired electron 2015 determined by the interactions of atoms and take up partial residence in a d orbital and molecules with each other is no better of a transition metal ion. These two mecha­ illustrated than examining the mechanis­ nisms dominate the chemistry of •NO, as tic foundations of the biological actions of described below. •NO. Because •NO has been studied since the dawn of chemistry and is one of the Pairing of the unpaired electron ten smallest stable molecules in Nature [2], One of the most important principles in understanding of its basic chemistry is rela­ chemistry is that electrons exist in atoms and tively well developed. The three properties molecules almost exclusively in pairs. This that most determine this chemistry are that means of course that the unpaired electron •NO is small, uncharged and (unlike virtu­ in •NO will require another unpaired elec­ ally all other molecules) contains an unpaired tron, which explains the rich chemistry of the part of

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the rate decreases exponentially with decreasing •NO O 2 (B) concentration, and at the nanomolar concentrations of (A)  NO2 ·NO2 •NO biologically (except perhaps under some inflam­ ·NO RSH matory conditions) this reaction is extremely slow and likely to be of very limited importance [4]. There how­ ONOO RSOH + NO  2 ever may be specific cellular microenvironments (spec­ O •- 2 ifically the hydrophobic phases of membranes and CO 2 lipid particles) where the reaction is accelerated due

to increased solubilities of both •NO and O2 in these phases [6]. A much more important route quantita­ •- - tively to biological formation of nitrite from •NO is an ·NO2 + CO3 NO3 (1/3) (2/3) ill-defined process(Figure 1B), whereby cells consume

•NO and O2 [7]. This reaction may well be the sec­ Figure 1. The preponderant covalent reactions of •NO ond most important biological reaction of •NO quan­ and progeny under biological conditions. Reactions titatively (second to reaction with oxyhemo­globin, not balanced chemically. (A) should be very minor vide infra). The mechanism(s) and intermediates are except at very high (•NO), but could be more important unknown. in specific microenvironments; product may not be ‘authentic’ •NO . (B) Mechanism(s) unknown; probably •NO also reacts with the one electron reduced form 2 •- second most preponderant biological •NO reaction of oxygen, superoxide (O2 ), and unlike the O2 reac­

(behind O2Hb reaction). tion is extremely rapid and kinetically first order with •- [O2 ]. In this case, the immediate product, peroxy­ reaction of •NO with other radical species. This also nitrite (ONOO-), is not a radical but it is a moderately explains why •NO is relatively unreactive to virtually strong two-electron oxidant. Under biological condi­ - all molecules in biology. These principles provide clar­ tions the major reaction of ONOO is with CO2. This ity to statements that •NO is ‘reactive’ or ‘unreactive.’ reaction produces a very transient species (nitroso­ - In reality, •NO does not react with almost all mol­ peroxocarbonate, ONOOCO2 ), 2/3 of which forms - ecules (in that sense being ‘unreactive’) but reaction the relatively unreactive nitrate anion (NO3 ) and also

with other radical molecules is very rapid (‘reactive’) regenerates CO2 and 1/3 rapidly homolyzes (breaks the because it involves the very favorable pairing of two peroxo O-O bond with each electron going to separate Figure 1 • unpaired electrons. diagrams the quantita­ products) to form two radicals, NO2 and carbonate •- tively most important biological reactions of •NO with radical anion (CO3 ). Thus, •NO essentially converts •- nonmetals and its subsequent reactive nitrogen species 1/3 of a relatively mild oxidant (O2 ) into two highly (RNS) [3]. oxidizing radicals. Under biological conditions another Among the biological molecules with unpaired elec­ reaction of peroxynitrite that occurs, although second

trons, molecular oxygen (O2) is pre-eminent. Although quantitatively to CO2, is with cellular thiols (RSH) to

O2 has an even total number of electrons, in the most result in a two-electron oxidation to form the moder­ common form (the ground state) two of the electrons ately reactive sulfenic acid (RSOH). Finally, in certain are unpaired (occupy separate molecular orbitals) and microenvironments protonated peroxynitrite (peroxy­ (Figure 1A) so the reaction of •NO with O2 is with­ nitrous acid, ONOOH; pKa = 6.8) homolyzes to • out doubt the single most studied reaction of •NO. produce NO2 and the very highly oxidizing hydroxyl However, in spite of this attention (extending back to radical (•OH). John Dalton, the ‘Father of Chemistry,’ who used it The overall ‘take home lesson’ is that the initial reac­

to perform the first determination of the 2O content tions of •NO with nonmetals under biological condi­ of the atmosphere [4]) there are many aspects of this tions results in two major effects, oxidation of cellu­ complex reaction that are not understood. The imme­ lar nucleophiles and formation of the relatively stable diate product is generally (although not universally [5]) nitrite and nitrate anions. The two major nucleophiles believed to be another small radical molecule, nitro­ that are best studied are cysteine thiol (proteins and • gen dioxide ( NO2). Although •NO is not an oxidant, also the small peptide glutathione, GSH) and the • NO2 definitely is and it reacts rapidly with many phenolic ring of tyrosine. One-electron oxidation • •- molecules ‘rich’ in electrons (nucleophiles) to extract by the radicals ( NO2, CO3 and, to a lesser extent, one electron, generating another radical and form­ •OH) generates the thiyl and tyrosyl radicals. These - ing nitrite anion (NO2 ) from the •NO2. This •NO/ can be considered ‘first generation’ radical products

O2 reaction exhibits unusual kinetics in that the rate because they, like •NO, become stabilized primarily is second order with •NO concentration. This means by reaction with other molecules with unpaired elec­

10.4155/FSO.15.59 Future Sci. OA (2015) 1(1), FSO59 future science group Nitric oxide: a brief overview of chemical & physical properties relevant to therapeutic applications Special Report trons. Thiyl radical reaction with •NO will result in reductions of •NO. In fact, undoubtedly the most nitrosothiol, a covalent compound containing the important reaction of •NO biologically (quan­ nitroso functional group (R-S-N = O). Tyrosyl radi­ titatively) is the extremely rapid reaction with cal reaction with •NO2 will result in nitrotyrosine, a oxyferrohemoglobin to form nitrate and ferriheme: covalent compound with the nitro functional group 2++3 : ++" - NO Fe ON23OFe Equation 1 (R-NO2). These are by far the two best studied nitro­ gen oxide adducts formed from •NO biologically. This electron transfer can also occur in reverse, with Another commonly cited mechanism for RSNO for­ electron flow to the ferriheme to form ferroheme; mation involves the reaction of •NO2 with •NO to this is the basis for the formation of •NO from yield nitrous an­hydride (N2O3) which reacts with nitrite, which is in the same formal oxidation state as + thiol (and also H2O) by transfer of the nitroso group oxidized •NO, nitrosonium(NO ; nitrosonium will (transnitro­sation). Although this reaction dominates never exist as a free ion under biological conditions the chemical reactivity of •NO in pure solution under because of extremely rapid reaction with H2O): aerobic conditions, it is almost certainly unimportant ++23+ - - ++ " : ++ biologically (except perhaps in specific microenviron­ NO2 HFeNOFeOH Equation 2 ments [6]) because it is much more likely that the •NO2 It is important to note that any mechanism that will react by one-electron oxidation with nucleophile results in formation of •NO from nitrite will always (e.g., GSH which is present in cells at up to 10 mM involve consumption of a proton and so commonly concentrations) than to react with the nanomolar will be more rapid as the pH is lowered. This is ­concentrations of •NO [8]). true also when transition metal is not involved, for example, the well known reactions that occur upon Partial residence in transition metal d orbitals acidification of nitrite involving nitrous anhydride

Transition metals are so named because in the peri­ (N2O3) [9]: odic table they represent ‘transition’ elements between Equation 3 2 22) + 23 the s and p orbitals, with electrons (both paired and HNO HO NO unpaired) occupying d orbitals. These orbitals can Equation 4 23)::+ 2 accommodate up to 10 electrons and in general can NO NO NO be fairly closely spaced in energy, depending on the The net effect of this reaction sequence is electron arrangement of ligands around the metal. These prop­ transfer from one nitrite to another, forming •NO2 erties mean that transition metals are excellent ‘homes’ (the one-electron oxidized product of nitrite) and to unpaired electrons. However, although complete •NO (the one-electron reduced product of nitrite). electron transfer from a radical can occur, it is more There also are proposals involving a reaction common that the unpaired electron(s) is shared by the sequence associated with both heme iron oxidation- radical molecule and the transition metal. Sharing of reduction and pairing-unpairing of electrons in electrons by atoms and molecules is of course called a nitrogen oxides [10,11] . The biological conundrum

‘bond’ so in particular •NO and O2 both form bonds these schemes are meant to address is how the vaso­ to transition metals. The bond that is formed is dis­ dilatory activity of nitrite, apparently attributable tinct from an organic covalent bond and is called a to reduction to •NO by deoxyhemoglobin, is pre­ coordinate bond. served when the •NO is produced within the red The best studied transition metal which bonds •NO cell, an environment containing massive concentra­ is iron, which bonds in either the ferrous (Fe2+) or ferric tions of an extremely rapid scavenger (oxyhemoglo­ (Fe3+) state. The dissociation of •NO from ferroheme bin, even in relatively hypoxic areas). The essential proteins is in general much slower than for ferriheme feature of these schemes is the notion that the •NO proteins, so •NO is bound much more tightly to ferro­ is ‘shielded’ from scavenging in the form of N2O3, heme. Since the coordinate bond is so different from which is produced by hemoglobin-catalyzed anhy­ the covalent bond there are different terminologies for dration (loss of water) from two molecules of nitrite. the nitrogen oxide, nitroso for the functional group in The N2O3 could escape the red cell and homolyze, covalent compounds and nitrosyl for the ligand bound forming •NO (and •NO2) and thus inducing dila­ to transition metal. tion. The overall nitrogen oxide chemistry (reactants and products) is identical to formation of •NO from Reactions involving both pairing of unpaired acid nitrite (Equation 3). The two mechanisms that electrons & transition metals have been proposed for deoxyhemoglobin catalysis Because the iron in many hemoproteins is redox are illustrated in Figure 2, denoted by ‘A’ [10] and active it can participate in one-electron oxidation- ‘B’ [11] .

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of the ferroheme complex and is termed ‘reductive Fe2+ nitrosylation’. The mechanism involves the oxidation + H - of •NO to a nitrosating species which can accom­ NO2 plish the nitrosation of nitrite to form N2O3. There is at present no consensus as to which mechanism OH- • NO ‘A’ or ‘B’ is responsible for the formation of N2O3 from nitrite and it is quite possible that both occur at {FeNO}6 Fe3+ appreciable rates.

Physical properties of •NO: a lone molecule NO - NO - 2 2 in search of a sink A B The metaphor of a molecule involved in a ‘search’ for a target is useful as a didactic teaching tool, but N O 2 3 it suffers from the serious caveat that it is fallaciously anthropomorphic. That is, molecules are of course 2+ Fe3+NO - Fe 2 nonsentient and in solution neither travel in linear paths nor purposely pursue a specific direction (the N O ·NO 2 3 common usage (including by the author) of straight­ lines in schematic diagrams unfortunately tends to exacerbate this misconception). The movement of Figure 2. Possible mechanisms for formation of N2O3 by reaction of nitrite with deoxyhemoglobin. Two •NO is by random diffusion, meaning the probabil­ mechanisms are denoted by ‘A’ and ‘B,’ although both ity of displacement in any direction is equal to the may occur at appreciable rates. probability in any other direction. Even though an individual molecule is equally Consideration of this mechanism illustrates the likely to move in any direction, collections of mol­ utility of the Enemark-Feltham notation for des­ ecules can certainly move in a certain direction. This ignation of iron-nitrosyl complexes [12], which is will occur if there are concentration gradients and y {Fe(NO)x} where ‘x’ denotes the number of nitrosyls it is a result of the fact that the probability of move­ coordinated to the iron and ‘y’ is the number of elec­ ment between two compartments with different con­ trons in the d orbitals of the iron plus the number centrations is greater for movement into the location of unpaired (π*) electrons from the •NO(s). In this with lower concentration and the result is that net case, the iron-nitrosyl is formed from nitrite binding movement will occur down a gradient. Thus, a col­ to a ferrous iron, accompanied by uptake of a pro­ lection of •NO molecules will always move away from ton and loss of hydroxide (anhydration). Since the the point at which it is made and in the direction of a nitrogen oxide in this case is formally one-electron sink. Since •NO is a very small and uncharged mol­ oxidized •NO (protonated nitrite can be considered ecule it has a high diffusion constant and will travel + - formally as nitrosonium hydroxide, [NO ][OH ]), great distances in a very short time [15] . However, this complex is thus designated {FeNO}6 because the presence of a rapid sink will greatly confine its no unpaired electron is contributed by the nitro­ diffusion field if located in ­proximity to the source. gen oxide and the iron contains six d electrons. The identical complex can also be formed from binding Financial & competing interests disclosure of •NO (one unpaired electron) to ferric iron (five This work was funded in part by NIH grant CA-131653. unpaired electrons). According to molecular orbital The authors have no other relevant affiliations or financial theory the chemical identity of the complex is the involvement with any organization or entity with a finan- same ({FeNO}6) no matter which mechanism is cial interest in or financial conflict with the subject matter responsible for formation; in essence, it makes no or materials discussed in the manuscript apart from those difference where the electrons were located in the ­d i s c l o s e d . reactants because in the complex they are all shared No writing assistance was utilized in the production of by both the iron and the nitrogen oxide (the very this manuscript. ­definition of ‘molecular orbitals’). Much of the basis of these proposals is derived Open access from work by Ford [13] on the mechanism of a phe­ This work is licensed under the Creative Commons Attribu- nomenon first reported in 1937 [14] that addition of tion 4.0 License. To view a copy of this license, visit http:// •NO to ferriheme proteins results in the formation creativecommons.org/licenses/by/4.0/

10.4155/FSO.15.59 Future Sci. OA (2015) 1(1), FSO59 future science group Nitric oxide: a brief overview of chemical & physical properties relevant to therapeutic applications Special Report

Executive summary • The chemistry of •NO is determined by the fact that it is a radical (contains an unpaired electron). • •NO reacts at appreciable rates with only two molecular targets, molecules that possess unpaired electrons and transition metals. • The chemistry of •NO is dominated by oxidation-reduction reactions, which can produce other nitrogen oxide species with much higher reactivity that •NO itself. • •NO binds avidly to transition metals and can undergo further oxidation-reduction and bond formation/ breakage. • •NO is highly diffusible in the biological milieu.

References 8 Lancaster JR Jr. Protein cysteine thiol nitrosation: maker or marker of reactive nitrogen species-induced nonerythroid 1 Raju TN. The Nobel chronicles. Robert Francis Furchgott cellular signaling? Nitric Oxide 19, 68–72 (2008). (b 1911), Louis J Ignarro (b 1941), and Ferid Murad (b 1936), Lancet 356(2000), 346 (1998). 9 Butler AR, Ridd JH. Formation of nitric oxide from nitrous acid in ischemic tissue and skin. Nitric Oxide 10 20–24 2 Lancaster JR Jr. Nitric Oxide in Cells. Am. Sci. 80, 248–259 (2004). (1992). 10 Robinson JM, Lancaster JR Jr. Hemoglobin-mediated, 3 Lancaster JR Jr. Nitroxidative, nitrosative, and nitrative hypoxia-induced vasodilation via nitric oxide: mechanism(s) stress: kinetic predictions of reactive nitrogen species and physiologic versus pathophysiologic relevance. Am. J. chemistry under biological conditions. Chem. Res. Toxicol. Respir. Cell Mol. Biol. 32 257–261 (2005). 19, 1160–1174 (2006). 11 Basu S, Grubina R, Huang J et al. Catalytic generation 4 Li Q, Lancaster JR Jr. A conspectus of cellular mechanisms of of N O by the concerted nitrite reductase and nitrosothiol formation from nitric oxide. For. Immunopathol. 2 3 anhydrase activity of hemoglobin, Nat. Chem Biol. 3 Dis. Therap. 3, 183–191 (2012). 785–794 (2007). 5 Wink DA, Darbyshire JF, Nims RW, Saavedra JE, Ford 12 Enemark JH, Feltham RD. Principles of structure, bonding, PC. Reactions of the bioregulatory agent nitric oxide in and reactivity for metal nitrosyl complexes. Coord. Chem. oxygenated aqueous media: determination of the kinetics for Rev. 13, 339–406 (1974). oxidation and nitrosation by intermediates generated in the 13 Ford PC, Fernandez BO, Lim MD. Mechanisms of reductive NO/O2 reaction. Chem. Res. Toxicol. 6, 23–27 (1993). nitrosylation in iron and copper models relevant to biological 6 Moller MN, Li Q, Lancaster JR Jr, Denicola A. Acceleration systems. Chem. Rev. 105, 2439–2455 (2005). of nitric oxide autoxidation and nitrosation by membranes. IUBMB Life 59, 243–248 (2007). 14 Keilin D, Hartree EF. Reaction of Nitric oxide with haemoglobin and methaemoglobin. Nature 139, 548 7 Thomas DD, Liu X, Kantrow SP, LancasterJRJr. The (1937). biological lifetime of nitric oxide: implications for the 15 Lancaster JR Jr. A Tutorial on the diffusibility and reactivity perivascular dynamics of NO and O2. Proc. Natl Acad. Sci. USA 98, 355–360 (2001). of free nitric oxide. Nitric Oxide 1, 18–30 (1997).

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