An international journal of inorganic chemistry
1477- 9226( 2010) 39: 22; 1- 7
King et al.
Oxidative heme protein-mediated
nitroxyl (HNO) generation
Dilworth et al.
One and two photon fluorescent
complexes of rhenium and their
Volume 39 | Number 22 | 14 June 2010 | Pages 5189–5400
PERSPECTIVE www.rsc.org/dalton | Dalton Transactions
Oxidative heme protein-mediated nitroxyl (HNO) generation
Julie A. Reisz, Erika Bechtold and S. Bruce King*
Received 15th January 2010, Accepted 25th February 2010
First published as an Advance Article on the web 20th March 2010
The distinct biological properties of nitroxyl (HNO) have focused research regarding the chemistry and
biology of this redox relative of nitric oxide (NO). Much of HNO’s biological activity appears to arise
through modification of thiol-containing enzymes and proteins and reactions with iron-heme proteins.
The reactions of HNO with hemoglobin and myoglobin serve as a general model for understanding
HNO reactivity with other heme proteins. Interaction of HNO with catalase and soluble guanylate
cyclase may have biological roles. While endogenous HNO formation remains to be described, we
summarize work that reveals HNO formation through oxidative heme protein metabolism of various
nitrogen-containing substrates including hydroxylamine, hydroxyurea, hydroxamic acids, cyanamide,
and sodium azide. Depending on the enzyme, the nascent HNO reductively nitrosylates the heme
protein or escapes the heme pocket as HNO. Such results define an alternative metabolism-based route
to HNO that may inform endogenous HNO production.
The establishment of nitric oxide (NO) as an important mediator
to other nitrogen oxides as NO sources or independent biological
signaling agents.1Studies from numerous laboratories over the
last 20 years show that nitroxyl (HNO), a structurally similar
compound related to NO by one-electron reduction and proto-
nation, elicits biological and pharmacological activities distinct
from NO. These findings have driven work to better understand
the chemistry and reactivity of HNO, to discover alternative
methods of HNO generation and detection, and to develop HNO
as a therapeutic entity. A number of excellent recent reviews
detail HNO chemistry, biochemistry, biology, and therapeutic
potential.2-5This perspective will briefly review HNO biology and
Department of Chemistry, Wake Forest University, Winston-Salem, NC,
27109, USA. E-mail: email@example.com; Fax: 336-758-4656; Tel: 336-758-
Julie A. Reisz
The author received a BS in
Chemistry from Allegheny Col-
lege in 2007. She is currently a
istry at Wake Forest University
working under the direction of S.
Bruce King. Her current research
involves phosphine-mediated re-
actions of HNO in effort to iden-
tify and develop new methods
to detect and quantify HNO in
The author received a BS de-
gree in Chemistry from Virginia
Polytechnic Institute and State
University in 2006. She is now
pursuing a PhD in Chemistry at
direction of S. Bruce King. Her
current research includes using
ate protein labels for the trapping
and detection of cysteine-based
in cells, specifically sulfenic acids
and S-nitrosothiols. The author
plans on pursuing postdoctoral work in the field of biological
chemistry after graduation.
chemistry, summarize the known reactions of HNO with heme
proteins, and examine HNO formation by heme protein-mediated
oxidation of various substrates.
Nitroxyl (HNO) releasing compounds consistently display bio-
logical actions that often differ from NO or NO donors and
these activities have been reviewed in detail.2,3As nitroxyl rapidly
oxide, biological studies of HNO rely upon donor compounds.5-8
Angeli’s salt (AS, Na2N2O3) spontaneously decomposes at neutral
pH to HNO and nitrite and represents the most common
HNO source for most chemical and biological studies.5,9This
requirement of HNO donors highlights the importance of the
development of alternative HNO sources.
and this activity forms the basis for the use of cyanamide as
This journal is © The Royal Society of Chemistry 2010Dalton Trans., 2010, 39, 5203–5212 | 5203
a treatment for alcoholism.10,11Nitroxyl releasing compounds
exhibit numerous cardiovascular effects including vasorelaxation,
an action similar to NO releasing compounds.12Angeli’s salt
enhances myocardial contractility by increasing calcium cycling
and by sensitizing myocardial responsiveness to calcium, leading
to the suggestion of HNO as a new therapy for congestive heart
failure.13-16Nitroxyl donors demonstrate a different pattern of
protection/exacerbation during myocardial ischemia/reperfusion
injury compared to NO donors, further distinguishing the biolog-
ical activities of these redox-related molecules.17HNO reacts with
hemoglobin to decrease the amount of NO scavenging by plasma
hemoglobin suggesting HNO as a possible therapy for various
this activity forms the basis of potential HNO-based cancer
therapies.19-21In the presence of oxygen, nitroxyl donors damage
DNA in a pattern different from NO donors and Angeli’s salt
generates hydroxyl radicals under some conditions.22,23HNO
affects the nervous system by modulating the NMDA receptor,
and Angeli’s salt enhances ischemic cerebral injury and oxidative
neurotoxicity.24,25In general, the molecular mechanistic basis of
these wide ranging activities appears to arise from HNO’s interac-
tion with biologically relevant 1) thiol-containing proteins and 2)
oxides including NO.
1.2 Nitroxyl chemistry
nitroxyl predominantly exists in its protonated form (HNO) in
aqueous solution at physiological pH with an approximate pKaof
11.4.26-28Thenitroxylanion(-NO) possessesatriplet groundstate,
similar to oxygen, kinetically retarding HNO deprotonation.27-29
The relatively high pKacoupled with the thermodynamic unfa-
vorability of NO to
-NO reduction permits HNO to exist as a
S. Bruce King
The author earned BS and MS
degrees in forestry and phar-
maceutical science from West
Virginia University in 1985 and
1988, respectively. He received
his PhD in organic chemistry
from Cornell University in 1993
under the direction of Bruce
Ganem. Following a postdoctoral
fellowship with K. Barry Sharp-
less at the Scripps Research In-
stitute, he joined the faculty of
sity where he is now a professor.
The author’s main research interests include the development and
chemistry of new nitric oxide and nitroxyl donors, the reactions of
defined species in biological systems, especially in the absence of
Similar to C-nitroso compounds and lower molecular weight
aldehydes (formaldehyde), nitroxyl dimerizes to hyponitrous acid
that dehydrates to nitrous oxide (Scheme 1).6Theoretical studies
predict favorable thermodynamic parameters for the reaction
of HNO with softer nitrogen and sulfur (compared to oxygen)
nucleophiles.27Experimentally, HNO acts as an electrophile and
readily reacts with amines, thiols, and phosphines to generate di-
azenes, disulfides or sulfinamides, and aza-ylides, respectively.30-32
Scheme 1 depicts the formation of the products during the
reaction of HNO with thiols through the intermediacy of an
N-hydroxysulfenamide that reacts further to give disulfide or
rearranges to sulfinamide. Such reactivity likely mediates a large
portion of HNO’s thiol-dependent biological activity.
Electrophilic reactions of HNO.
The weak N–H bond strength of HNO allows it to act as
an H-atom donor and reducing agent (forming NO) and HNO
quenches nitroxide radicals such as TEMPO and polyunsaturated
fatty acid radicals.33-35Nitroxyl directly reacts with molecular
oxygen although the kinetics, products, and mechanism of this
nitrogen and oxygen allow HNO to interact with various Lewis
acids, particularly metals. Similar to NO, HNO exhibits diverse
chemistry with various metals and metal-containing proteins and
the next section of this perspective summarizes the reactions of
HNO with metal heme-containing proteins.
2.Reactions with heme proteins
Reactions of NO and ferrous and ferric myoglobin (Mb) and
hemoglobin (Hb) are well-characterized, play numerous physio-
logical roles, and require consideration before discussing HNO-
heme protein reactions. Oxygenated ferrous Mb and Hb serve as
and nitrate with a rate constant of 5-8 ¥ 107M-1s-1for MbO2and
9 ¥ 107M-1s-1for HbO2(Scheme 2).37,38Deoxygenated Hb or Mb
reacts with NO producing ferrous nitrosylated heme at a nearly
identical rate (3 ¥ 107M-1s-1for Hb, Scheme 2).39Ferric Hb or
Mb in the presence of two NO equivalents undergoes reductive
nitrosylation forming the ferrous nitrosyl heme and nitrite with
an associated pseudo-first order rate constant ranging from 104-
107M-1s-1for metMb.40-42Determining the rate for the reaction of
Reactions of hemoglobin and myoglobin with NO.
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metHb with NO is complicated by heme cooperativity, resulting
in a slow phase (k = 5 ¥ 103M-1s-1) and a fast phase (k = 1.4 ¥
104M-1s-1) which give different observed rates.41(Scheme 2).
Although the reactions between HNO and heme proteins
remain less studied than the reactions between NO and heme
proteins, the reactions/interaction of HNO with hemoglobin and
myoglobin provide well-characterized examples for comparison.
2.1 Oxyhemoglobin & oxymyoglobin
The oxygenated ferrous hemes of myoglobin and hemoglobin
undergo rapid oxidation to the ferric forms upon reaction with
HNO generated from Angeli’s salt (AS). Studies with MbO2
suggested the reaction to be first order in AS and zero order in
heme protein.43In addition to ferric heme formation, this reaction
also yields nitrate possibly via H-atom abstraction of HNO
to NO with the heme-bound oxygen being converted to HO2
heme provides additional ferric heme and NO3
original proposal for nitrate formation,31neither NO or HO2
generation have been substantiated during this reaction and other
mechanisms including the hydroxylamine radical also explain the
observed products.5Kinetic studies support this mechanism with
a 2:1 (heme:HNO) stoichiometry and illustrate a role for NO
Reaction of oxyHb or Mb with HNO.
The reaction of HNO with MbO2is among the most rapid of
the known traps for HNO with a derived rate constant of 1 ¥
107M-1s-1.35MbO2readily oxidizes HNO to NO, which may have
important biological implications for endogenous NO generation
MbO2prevents the use of metMb formation as a marker for HNO
release by donor compounds or as an indirect method of in vivo
2.2Deoxyhemoglobin & deoxymyoglobin
Deoxygenated ferrous hemes directly react with HNO to form
coordination complexes and Farmer has recently reviewed HNO
as a heme ligand (Scheme 4).44Early studies showed the initial
reaction of deoxyHb and AS provides HbNO and metHb in
with initial metHb formation followed by HNO-promoted metHb
reductive nitrosylation to HbNO in the second.43,45The overall
reaction mechanism appeared to be second order in AS and
zero order in deoxy heme.45Ultimately, Doyle suggested that
the reaction of deoxyMb with AS produces an intermediate
Reaction of deoxyHb or Mb with HNO.
ferrous Mb(HNO) complex, which reacts with NO2
to metMb and MbNO as observed.31The reactivity and the
its detection and complicated the interpretation of these initial
The complexation of HNO with ferrous deoxyhemes remained
largely under-appreciated until Farmer demonstrated the reduc-
tion of MbNO using CrIIreagents in aqueous alkaline solution
provides a ferrous Mb(HNO) complex.46Remarkably, the HNO
adduct of deoxyMb is stable in anaerobic solution with a half-
life of several months making deoxyHb trapping of HNO es-
sentially irreversible. Electrochemical methods oxidize the ferrous
Mb(HNO) adduct to MbNO over several minutes.46
Purified aqueous samples of ferrous Mb(HNO) are stable over
pH 6–10, and as a result, the heme pocket of the Mb(HNO)
2D-NMR studies of the ferrous Mb(HNO) complex reveal a
hydrogen bond between the HNO proton (15 ppm) and the
distal pocket histidine residue (His64) analogous to the hydrogen
bonding in MbO2known to confer significant stability to oxygen-
complex formation is estimated at 1.4 ¥ 104M-1s-1.48The ferrous
Mb(HNO) complex reacts with AS-derived NO2
metMb and NO, which subsequently form MbNO. (Scheme 4).
Exposure of the ferrous Mb(HNO) complex to air, NO2
NO leads to oxidation to metMb over several minutes.48Most
of human, soy, and clam hemoglobins that may be utilized as
heme pocket models for probing oxygen binding parameters. In
contrast to oxyhemes, HNO-bound hemes offer characteristic
NMR signals (for1H and15N) distinct for each heme, a valuable
-, giving rise
2.3Methemoglobin & metmyoglobin
The heme of methemoglobin (metHb) and metmyoglobin
(metMb) is a paramagnetic FeIII
complex.50,51In the presence of AS-derived HNO, the ferric heme
undergoes reductive nitrosylation, producing a ferrous-nitrosyl
complex (FeIINO, Scheme 5).5,31,52,53Mechanistic studies suggest a
direct complexation between HNO and FeIIIfollowed by electron
5-coordinate high spin
Reaction of metMb with HNO.
Reductive nitrosylation by AS proceeds with a rate constant of
8 ¥ 105M-1s-1for metMb and is zero order in metMb and first
10-4M-1s-1, AS decomposition to HNO and NO2
slower than the rate of formation of Mb(FeII)NO.53Determining
the rate of reductive nitrosylation of metHb is complicated by
the reaction of HNO with the b-93 cysteines of Hb.31Monitoring
the reaction progress by measuring HbNO formation showed an
initial “slow phase” followed by rapid conversion to the nitrosyl.31
Doyle demonstrated that the chemical modification of the b-
93 sulfhydryl groups to the thioacetamide derivative completely
This journal is © The Royal Society of Chemistry 2010Dalton Trans., 2010, 39, 5203–5212 | 5205
eliminated the initial slow phase for HbNO formation and the
observed rate was consistent to that of metMb with AS.31
Several ideas exist concerning the possible biological implica-
in resonance with FeIIINO-and while not currently substantiated
has been proposed as a possible endogenous source of HNO
(Scheme 6).55,56FeIINO could also act as a source of NO,
however the dissociation of nitric oxide from FeIINO is very
slow with 10-3s-1reported as the fastest rate constant.57,58Under
physiological conditions, the release of NO from FeIINO may first
require oxidation to FeIIINO as this species has a rate constant for
NO release that is about 1000 times higher.41
Iron-nitrosyl complexes as HNO or NO sources.
Five-coordinate ferric heme proteins such as metHb or metMb
have been proposed as efficient HNO traps for HNO. The ferrous
nitrosyl products have unique UV/Vis absorption and EPR
spectra that can be monitored as an indirect measure of [HNO]
in samples.45This method for detecting HNO remains impractical
in vivo due to the fact that ferric hemes also react with NO to give
NO and HNO problematic.58-60
2.4 Horseradish peroxidase
The resting state of the peroxidase heme, including horseradish
peroxidase (HRP), is a 5-coordinate high spin ferric complex
with a proximal histidine ligand.61The HRP catalytic cycle
commences with binding of H2O2to the ferric heme, oxidation to
a ferryl (Fe4+) intermediate (Compound I), and subsequent two-
Compound II) with overall reduction of H2O2 to water.61As
expected for a pentacoordinate ferric heme protein like metHb,
HRP forms a ferrous nitrosyl complex upon HNO exposure
(Scheme 7).35,62,63An estimated rate constant of 2 ¥ 106M-1s-1
complex undergoes auto-oxidation in the presence of O2to give
nitrate and regenerate ferric heme (Scheme 7).60,63,64
Reaction of HRP with HNO.
In addition to their antioxidant and protective effects,
peroxidases catalyze the conversion of hydroxylamine and hy-
droxyurea (NH2OH and NH2CONHOH) to HNO, suggesting
possible endogenous sources of HNO that will be discussed in
Catalase (cat) catalyzes the conversion of H2O2to H2O and O2in
a dismutation reaction.65,66Cat is also a 5-coordinate ferric heme
protein that reacts with HNO to form a ferrous nitrosyl complex
with an estimated rate constant of 3 ¥ 105M-1s-1(Scheme 8).35,64,67
Reaction of cat with HNO.
Similar to other ferrous nitrosyl complexes, this catalase complex
demonstrates excellent stability under anaerobic conditions, but
exposure of cat(FeII)-NO to O2 results in regeneration of the
ferric heme and nitrate (Scheme 8).67Ferric catalase resists reduc-
tion (sodium dithionite does not reduce ferric catalase) making
examples of ferrous catalase complexes rare and only prepared
through photochemical reduction sequences.42,68,69HNO acts as
an effective reductant for ferric catalase and yields cat(FeII)NO
making AS the reagent of choice for preparation.67Unlike HNO,
nitric oxide does not reductively nitrosylate catalase and this
reactivity difference between HNO and NO with ferric catalase
presents an opportunity to differentiate these redox species.
The preference of the catalase heme iron to remain in the ferric
state suggests the possibility of weak ligand binding to the ferrous
nitrosyl complex oxidizes oxyHb presumably through NO release
(Scheme 8).67Catalase mediated oxidation of hydroxylamine also
generates cat(FeII)NO that activates soluble guanylate cyclase
(sGC) presumably through NO exchange (Scheme 8).70,71The
ability of the cat(FeII)NO complex to act as an NO donor
may signify an important role for catalase in HNO physiology.
Catalase also catalyzes the formation of HNO from hydroxyurea,
cyanamide, and sodium azide (NaN3), and these transformations
will be addressed in Section 3.72-74
2.5 Cytochrome c oxidase
Cytochrome c oxidase (cyt c) is a membrane-bound copper-heme
during electron transport.75The heme pocket (which binds O2
during normal catalysis) is a high spin 5-coordinate ferric center
similar to metMb.75NO regulates cyt c activity by binding
and coordinating at the heme center, inhibiting mitochondrial
The reaction of HNO with cyt c(FeIII) gives cyt c(FeII) and NO
with an estimated rate constant of 4 ¥ 105M-1s-1(Scheme 9).35,77
This reaction most likely proceeds through the direct formation
of the iron nitrosyl complex that rapidly dissociates to FeIIand
NO, but this reaction may occur through an outer-sphere electron
transfer process.31,35,77,78Kinetic evaluation of a non-homologous
E. coli cytochrome d oxidase indicates the reaction is first order in
HNO and cyt d and involves direct interaction between HNO and
Reaction of cyt c with HNO.
The addition of AS to purified mitrochondria in the oxidized
However, when AS was incubated with SMPs (submitochondrial
particles) devoid of cyt c, a similar NO detection profile was
5206 | Dalton Trans., 2010, 39, 5203–5212This journal is © The Royal Society of Chemistry 2010
observed, indicating that an additional component in the inner
membrane of mitrochondria may also be responsible for the
oxidation of HNO.76
2.6 Soluble guanylate cyclase
Vasorelaxation, one of the most prominent physiological roles
of NO, occurs through activation of the heme protein soluble
guanylate cyclase (sGC), which catalyzes the conversion of GTP
to cGMP.80The vasodilatory mechanism involves binding of NO
Angeli’s salt elicits vasodilation of rabbit thoracic aorta and
bovine intrapulmonary artery at concentrations 100 times lower
than that of nitrite-mediated vasodilation.12As nitrite is also
an AS decomposition product, these results reveal the potency
of HNO.12Levels of cGMP were markedly increased following
exposure to AS, suggesting HNO-mediated activation of sGC.12
Additionally, relaxation of rat vasculature by AS is attenu-
ated by sGC inhibitor 1H-[1,2,4]oxadiazolo[4,3-a]quinoxalin-1-
one (ODQ).83Concentration-dependent vasorelaxation of rat
mesenteric arteries by AS occurs in the presence of NO-
oxyl-3-oxide (carboxy-PTIO) indicating that these effects do not
result from HNO oxidation to NO.84Overall, these results suggest
that HNO itself elicits vasodilatory effects through an sGC-
A recent study of bovine lung sGC activation by AS and
the structurally distinct HNO-donor 1-nitrosocyclohexyl triflu-
oroacetate (NCTFA) validates direct enzyme activation by HNO
(without HNO conversion to NO). In this work, AS and NCTFA
enhance the activity of sGC 20- and 60-fold, respectively.85
Removal of the heme retains basal enzyme activity but signifi-
cantly diminishes activation by HNO donors, implying that the
mechanism of HNO activation involves direct heme binding.85
Ferricyanide oxidation provided ferric heme sGC that proved
inactive toward both NO and HNO donors.85Reduction of
the enzyme then restored sensitivity to activation by both NO
and HNO donors. Given that HNO is not expected to form
sGC(FeII)NO from the reaction with ferrous sGC, the prevailing
proposed activation mechanism involves direct interaction of
(Scheme 10).85Interestingly, inhibition of sGC occurs at higher
concentrations of AS (100 mM) likely via reaction of excess HNO
with protein thiols.85Thus, HNO may provide a concentration-
and heme-dependent regulation of sGC activity.
Possible activation of sGC by HNO.
Trapping of HNO by the ferrous sGC heme may provide
an sGC(FeII)HNO adduct similar to complexes for hemoglobin
and myoglobin, with activity comparable to the sGC(FeII)NO
heme (Scheme 10). Another explanation for HNO-mediated sGC
reaction of ferrous sGC and HNO by an unknown mechanism or
in vivo oxidation of the FeIIHNO. A subsequent report challenges
these conclusions, asserting that HNO activation of sGC first
involves oxidation to NO, possibly by superoxide dismutase.86
examination as HNO offers promise as a potent vasorelaxant. Rat
aortic tissue does not build tolerance towards AS, an impediment
to long-term therapy with other nitrovasodilators.87
Cytochromes P450 (CYP450) represent a diverse group of heme
isoenzymes that serve as ubiquitous oxidizing agents. In mam-
malian systems, CYP450 participate in the biosynthesis of regu-
latory compounds such as steroids, prostaglandins, and fatty acid
derivatives and the metabolism of drugs and other xenobiotics.88,89
In the resting state, CYP450 exists as a pentacoordinate
ferric heme with a catalytic cycle that includes ferrous,
ferryl, and oxygen-bound complexes. The NO donors DEA/
NO ([Et2NN(O)NO]Na) and S-nitroso-N-acetylpenicillamine
(SNAP) potently inhibit CYP450 via several distinct mecha-
nisms.90Nitrosylation of the deoxygenated ferrous heme impedes
binding of molecular oxygen via reversible inhibition.90Binding
of NO to the heme may also displace the cysteine ligand, allowing
the catalytic cycle (Scheme 11).90,91
Inhibition of CYP450 by NO.
Treatment of S9 microsomes with AS increased inhibition of
CYP450 comparable to results obtained with SNAP.52Removal
of the HNO donor regenerates the active enzyme suggesting that
both NO and HNO reversibly regulate CYP450 activity.52The
mechanism of HNO inhibition involves a reductive nitrosylation
of the ferric heme resting state, providing the expected ferrous
nitrosyl complex formed when NO binds to the deoxy ferrous
heme (Scheme 12).52
Inhibition of CYP450 by HNO.
3. Oxidative heme protein-mediated HNO formation
Despite the recent interest in HNO biology, chemistry, and po-
tential therapeutic use, endogenous HNO production eludes con-
clusive description.2,3The biochemical conversion of L-arginine
This journal is © The Royal Society of Chemistry 2010 Dalton Trans., 2010, 39, 5203–5212 | 5207
to L-N-hydroxyarginine to nitric oxide, which is catalyzed by
nitric oxide synthase (NOS), provides a potential HNO-forming
pathway as this sequence passes through the formal HNO
nitrogen oxidation state (N = -3, -1, +1, +2 for L-arginine, L-
N-hydroxyarginine, HNO, and NO, respectively). In the absence
of the redox co-factor tetrahydrobiopterin, NOS catalyzes the
formation of HNO from L-arginine giving credence to such a
route for endogenous HNO formation.92While the distinct and
a natural role for HNO, the reactivity of HNO and the lack of
A number of N-containing substrates in lower nitrogen oxida-
protein mediated oxidation with subsequent HNO formation.
hydroxamic acids, azide, and cyanamide will be summarized.
Reductive pathways of biological HNO formation from NO
exist but will not be covered.93Oxidative metabolism defines an
alternative pathway for HNOgeneration andmay provide clues to
mechanisms of endogenous HNO formation. Catalase-mediated
oxidative HNO production from cyanamide, a clinical therapy for
mediated oxidations may further react to give other products
including NO or a ferrous nitrosyl complex.
3.1 HNO from hydroxylamine
Hydroxylamine rapidly reacts with heme proteins including oxy,
deoxy, and metHb to produce NO.64,94-96These reactions generate
other nitrogenous products including nitrogen gas and ammonia
but do not clearly form HNO.97
Treatment of hydroxylamine with HRP in the presence of hy-
drogen peroxide generates HNO as determined by HNO trapping
with glutathione and HPLC/mass spectrometric identification
of glutathione sulfinamide (Scheme 13).63Gas chromatographic
identification of nitrous oxide in the headspace of this reaction
further confirms HNO formation.63In this system, hydrogen
peroxide activates HRP to the reactive intermediate Compound
I that oxidizes hydroxylamine to HNO (Scheme 13). This HNO
can reductively nitrosylate HRP(FeIII) to yield HRP(FeII)NO that
can be detected by EPR spectroscopy and that auto-oxidizes to
the ferric heme and nitrate (Path a, Scheme 13).63Alternatively,
HNO may escape the heme pocket and react with other trapping
agents (glutathione) or dimerize to form nitrous oxide (Path b,
sulfinamide or nitrous oxide under these conditions.63A survey
of various heme proteins reveals that those with a histidine
HRP catalyzed HNO formation from NH2OH.
proximal ligand (HRP, myeloperoxidase, Hb and Mb) preferen-
tially form glutathione sulfinamide from hydroxylamine giving
evidence for “free” HNO in this system.63Proteins with proximal
The facile peroxidase catalyzed oxidation of hydroxylamine to
HNO coupled with the rapid thiol-mediated reduction of HNO
to hydroxylamine forms a potential redox cycle between HNO
and NH2OH. (Schemes 1, 13, and 14).63Scheme 14 shows such
a cycle including HRP and hydrogen peroxide as oxidants and
glutathione as the reductant. By such a cycle, HNO and NH2OH,
regardless of source, could interconvert depending on local redox
status possibly playing a signaling or protective role (Scheme 14).
NH2OH–HNO redox cycle.
Hydroxylamine, in the presence of catalase and a hydrogen
EPR spectroscopic examination of this reaction reveals the
formation of the Cat(FeII)NO complex,70which forms during
the reaction of HNO and ferric catalase. Given the inability of
NO to reduce ferric catalase,42these results strongly imply initial
HNO formation followed by reductive nitrosylation. This ferrous
catalase nitrosyl complex acts as an NO donor and may transfer
NO to sGC leading to enzyme activation.67Such a system appears
to represent a unique HNO-derived sGC activator and indicates
HNO donor/catalase systems may be considered as NO donors.
3.2 HNO from hydroxyurea and hydroxamic acids
Hydroxyurea demonstrates a diverse pharmacological profile and
mains an important treatment for a number of myeloproliferative
disorders, especially chronic myelogenous leukemia.98Hydrox-
term study indicates that hydroxyurea therapy reduces mortality
40%.99Hydroxyurea also acts as a source of NO, which has
drawn considerable interest as a sickle cell disease treatment.100,101
Multiple lines of evidence indicate the in vivo conversion of
hydroxyurea to NO in sickle cell disease patients.102,103
Hydroxyurea reacts with oxy, deoxy, and metHb in vitro
to form iron nitrosyl hemoglobin but these reactions do not
occur fast enough to account for NO production observed in
patients undergoing sickle cell therapy.104,105To reconcile these
observations, alternative mechanisms of NO formation from
hydroxyurea have been considered including: 1) peroxidase or
catalase mediated formation of NO from hydroxyurea and 2)
NO production after hydrolysis of hydroxyurea to hydroxylamine.
In the presence of urease, which catalyzes the hydrolysis of
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hydroxyurea to hydroxylamine, incubation of hydroxyurea with
hemoglobin rapidly forms iron nitrosyl hemoglobin.106
In the presence of hydrogen peroxide, HRP catalyzes the rapid
formation of HNO and NO from hydroxyurea.62Scheme 15
NO formation as well as the observed nitroxide radical and acyl
nitroso intermediates during this reaction.62Hydrogen peroxide
oxidation of HRP produces the reactive intermediate Compound
I that acts as a one-electron oxidant of hydroxyurea to give
the nitroxide radical and Compound II.62Further one-electron
oxidation by Compound II yields the acyl nitroso compound
and resting ferric HRP (Scheme 15).62Acyl nitroso compound
ammonia, and HNO, which dimerizes and dehydrates to nitrous
oxide (Scheme 15).62Further single electron oxidation of HNO by
the ferric heme of HRP provides a route for NO production. As
reactive intermediates may also act as potential single electron
oxidants of HNO.107These results show that this HRP system
an acyl nitroso compound intermediate and identifies peroxidases
as potential oxidants for the in vivo conversion of hydroxyurea to
HNO and NO.
HRP catalyzed HNO formation from hydroxyurea.
Similar to HRP, catalase in the presence of a hydrogen peroxide
generating system converts hydroxyurea to HNO and NO.67
EPR spectroscopy shows that under these conditions, hydrox-
yurea and catalase produce the ferrous-NO catalase complex
by the appearance of the characteristic triplet signal at g =
1.99 in the EPR spectra.67Scheme 16 outlines the proposed
mechanism for HNO and cat(FeII)NO formation during the
reaction with hydroxyurea. Hydrogen peroxide activates ferric
catalase to Compound I and direct two-electron oxidation of
Cat catalyzed HNO formation from hydroxyurea.
hydroxyurea yields the acyl nitroso compound intermediate.67
These results and the previously described HRP experiments
compound. Unlike the reaction of hydroxyurea and HRP, gas
chromatographic headspace analysis does not show the presence
of nitrous oxide supporting ferric heme trapping of HNO.67In
addition, room temperature EPR measurements fail to identify
the nitroxide radical of hydroxyurea supporting a direct two-
electron oxidation.67Hydrolysis of the acyl nitroso compound
and ammonia.67Reductive nitrosylation of the ferric heme of
catalase by HNO gives the ferrous NO-catalase complex.
These results, combined with those from HRP, describe a
defined pathway of HNO formation from hydroxyurea and oxida-
tive ferric heme proteins. Two-electron oxidation of hydroxyurea
produces the acyl nitroso compound that hydrolyzes to HNO
and carbamic acid. Nitroxyl formed in these reactions can 1)
escape the heme pocket (HRP) or 2) reductively nitrosylate the
ferric heme to yield ferrous-NO complexes (Cat). The structural
act as NO donors capable of sGC activation. Such work identifies
acyl nitroso compounds as HNO donors and HNO/ferric heme
protein couples as NO sources depending on the protein.
Hydroxamic acids (RCONHOH) demonstrate diverse bio-
logical activities primarily based upon their metal chelating
properties.108These agents find use for iron overload and
cancer treatment with suberoylanilide hydroxamate (SAHA,
a histone deacetylase inhibitor) being approved for cancer
treatment.109Hydroxamic acids bear structural similarity to
hydroxyurea, and hydrolysis of these carboxylic acid derivatives
yields hydroxylamine.110While these properties make hydroxamic
little work in this area has appeared. A recent paper reports
treatment of SAHA with hydrogen peroxide and metMb produces
NO and the authors suggest the intermediacy of HNO but further
mechanistic characterization is not described (Scheme 17).111
NO formation from SAHA.
3.3HNO from cyanamide
Cyanamide (H2N-CN) has been used for many years to treat
alcoholism around the world.2Cyanamide elicits its effects by act-
ing as an HNO prodrug and requires bioactivation by catalase.11
dehydrogenase inhibiting normal ethanol metabolism.10Studies
with purified enzyme show both a reversible and irreversible
component to HNO inhibition of aldehyde dehydrogenase.10The
reversible portion of inhibition has been attributed to disulfide
formation and the irreversible inhibition from HNO-mediated
This journal is © The Royal Society of Chemistry 2010 Dalton Trans., 2010, 39, 5203–5212 | 5209
also forms the cyanide ion, evidence of cyanide toxicity in
cyanamide use has not been reported.2
Detailed chemical and biochemical studies outline the
mechanistic pathways of HNO release from cyanamide.10,11,112
Treatment of cyanamide with catalase and a hydrogen peroxide
generating system forms unstable N-hydroxycyanamide through
a unique catalase-mediated N-hydroxylation (Scheme 18).
N-Hydroxycyanamide can directly decompose to HNO and HCN
(Path a) or be further oxidized by catalase to nitrosyl cyanide
(ONCN, Path b).112Nitrile hydrolysis of nitrosyl cyanide gives
the same acyl nitroso species observed in the HRP and cat-
mediated oxidations of hydroxyurea and hydrolysis yields HNO
and carbamic acid that decomposes to carbon dioxide and am-
monia (Scheme 18).112Gas chromatographic/mass spectrometric
analysis and NMR spectroscopy provide evidence for HNO,
carbon dioxide, and cyanide formation in these studies.11,112The
identification of nitrous oxide and the modification of thiols
in aldehyde dehydrogenase clearly indicate the generation of
“free” HNO in this system and no evidence exists for HNO
trapping by the catalase ferric heme to give cat(FeII)NO.10,11,73
These results directly contrast those with the catalase/hydrogen
peroxide mediated oxidations of hydroxylamine and hydroxyurea
which form cat(FeII)NO but do not generate nitrous oxide.67One
may speculate a distinguishing role for the cyanide ion through
binding to ferric catalase and blocking reductive nitrosylation
allowing HNO diffusion. The chemical, biochemical, and clinical
body of work regarding cyanamide shows the feasibility of HNO
donors as therapeutic agents and supports the development of
other HNO donor systems.
Cat catalyzed HNO formation from cyanamide.
3.4HNO from sodium azide
Sodium azide has long been recognized to possess vasore-
laxant properties.94,113These actions occur through the acti-
vation of sGC via the intermediacy of NO, similar to other
metabolic activation to NO and the mixture of sodium azide,
catalase, and a hydrogen peroxide generating system forms
to directly reduce cat(FeIII) strongly implies HNO involvement in
the catalase-mediated oxidation of sodium azide.42As in the other
systems described, cat(FeII)NO appears to act as a NO donor
to that described for cyanamide may yield an N-hydroxyazide
intermediate that decomposes to HNO and nitrogen (Scheme 19),
which likely generates NO through the intermediacy of HNO and
a cat(FeII)NO. This system requires more study to distinguish
Proposed HNO formation from azide.
whether azide generates both NO and HNO or simply one of
the nitrogen monoxides.
Nitroxyl (HNO), one-electron reduced and protonated NO,
demonstrates biological activities distinct from nitric oxide (NO)
including the inhibition of alcohol metabolism, vasorelaxation,
enhanced cardiac muscle contractility, and the inhibition of
glycolysis prompting consideration of these HNO donors as
therapies for alcoholism, congestive heart failure, and cancer.
trophile with various biological nucleophiles (particularly sulfur-
containing proteins). Like NO, HNO also reacts with iron heme
Mb(FeII)HNO complex and reductively nitrosylates Mb(FeIII) to
involves the oxygen ligand to give Mb(FeIII) and NO. Currently,
these reactions with the common forms of Hb or Mb provide
a basis to understand HNO reactions with other heme proteins.
In general, other iron containing heme-proteins react with HNO
similar to Hb and Mb.
to be clearly defined. We summarize the oxidative heme protein-
mediated metabolism of various nitrogen substrates (hydroxy-
lamine, hydroxyurea, hydroxamic acids, cyanamide, and sodium
azide). Each of these compounds require oxidative activation by a
heme protein, generally cat or a peroxidase, to form HNO. These
processes generate higher oxidation state enzyme intermediates
(Compounds I and II) that convert the substrate to HNO or an
with the ferric heme to yield a ferrous nitrosyl complex or may
escape the heme pocket as HNO. Such work defines an alternative
oxidative metabolic mechanism of HNO formation compared to
strict chemical donors and may provide an insight into potential
endogenous HNO formation.
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