Ruthenium(III) readily abstracts NO from
L-arginine, the physiological
precursor to NO, in the presence of H2O2. A remarkably simple model
system for NO synthases
Celine J. Marmion,* Terry Murphy and Kevin B. Nolan*
Department of Chemistry, Royal College of Surgeons in Ireland, St. Stephen’s Green, Dublin 2, Ireland.
Received (in Cambridge, UK) 10th July 2001, Accepted 13th August 2001
First published as an Advance Article on the web 4th September 2001
Reaction of [Ru(Hedta)Cl]2 with H2O2 in the presence of
arginine, produces NO, in the form of an Ru(II)–(NO+)
complex and citrulline which is a remarkably simple model
system for the physiological NO synthase reaction.
removal of solvent, afforded a brown product† having an
intense n(NO) IR band at 1888 cm21 indicative of a linear,
diamagnetic Ru2+–NO+ containing complex.4,7‡ The product
of denitrosylation of arginine was shown by TLC and by mass
spectrometry to be citrulline, identical to the physiological
denitrosylation product, eqn. (1).§
The physiological importance of nitric oxide is now firmly
established.1 The processes in which it plays a pivotal role
include regulation of cardiovascular function, signalling be-
tween nerves in the peripheral and central nervous systems,
mediating host defence against bacteria and tumour cells and
many others.1 In living organisms NO is generated by oxidation
Ru(III) + arginine + H2O2 ? Ru(II)–NO+ + citrulline + H2O
(1)
A likely reaction mechanism involves initial hydroxylation of
arginine to N-hydroxyarginine by the green RuVNO complex¶
which is formed by oxidation of the ruthenium(III) complex with
hydrogen peroxide. This is remarkably similar to the first
monooxygenation step of the NO synthase reaction but involves
of the guanidine function of
involves two sequential monooxygenations and proceeds via
the stable intermediate N-hydroxy- -arginine ultimately afford-
ing -citrulline and NO. The entire process is catalysed by
L
-arginine, a reaction which
L
L
oxoruthenium( ) instead of a high valent oxoiron species and
V
haem-containing NO synthases and requires NADPH and O2.
Despite the physiological importance of NO the biosynthetic
pathway of this important reaction is not yet fully understood.
Analogous to the well established cytochrome P450 N-
oxidation chemistry, the first monoxygenation step is thought to
be mediated by a transient, high valent iron–oxo porphyrin
H2O2 as a surrogate active oxygen donor instead of NADPH and
O2. The oxidation of N-hydroxyarginine to citrulline and NO in
our system may occur by a ‘peroxide shunt’ mechanism similar
to that previously reported by Marletta and coworkers where
H2O2 supported oxidation of N-hydroxyarginine, in the pres-
ence of NO synthase, yields the same products.8 In our system
the Ru(III) complex fulfils the role of NO synthase as in the first
monooxygenation step. Alternatively in the presence of Ru(III),
NO abstraction from N-hydroxyarginine may occur by a
mechanism similar to that previously proposed for NO
abstraction from hydroxamic acids,5 involving hydroxylamine
(a known source of NO ligands)9 as an intermediate, and
analogous to an earlier mechanism proposed by DeMaster et al.
for the second NO synthase-mediated monooxygenation
step.10
In summary we have conclusively shown that in the presence
of hydrogen peroxide and [Ru(Hedta)Cl]2 arginine releases
NO and is converted into citrulline, a reaction identical to that
occurring physiologically. The precise structures of the ruthe-
nium complexes involved in the reaction sequence as well as the
reaction mechanism are currently being investigated.
complex, which hydroxylates -arginine and forms enzyme
L
bound N-hydroxyarginine.1,2 The proposed second mono-
oxygenation step involves nucleophilic attack by a peroxo
ligand attached to ferric haem on the guanidine carbon giving a
tetrahedral intermediate from which NO and citrulline are
eliminated. This step however, which has no known precedent
in biological chemistry, is much less understood than the first,1
and there are many unanswered questions relating to the fine
mechanistic detail of the reaction. The ability of ruthenium(III
)
complexes to form oxoruthenium( ) species with the potential
V
to hydroxylate substrates,3 such as arginine and the affinity of
the resulting reduced ruthenium(III) species for NO,4,5 prompted
us to explore systems such as these as models for NO synthase,
Fig. 1. One such system based on the well characterised
complex K[Ru(Hedta)Cl] and utilising H2O2 in place of
NADPH and O2 is described in this communication.
We thank Mr Brendan Harhen, Department of Clinical
Pharmacology, RCSI, for mass spectra, The Microanalytical
Laboratory, University College Dublin for microanalysis and
Enterprise Ireland, The Irish Government under its Programme
for Research in Third Level Institutions, RCSI and EU COST
D20 for financial support.
Notes and references
† Reaction of an aqueous solution of K[Ru(Hedta)Cl]·2H2O (400 mg, 0.80
mmol) with an aqueous solution of L-arginine (557 mg, 3.2 mmol) in the
presence of an excess of H2O2 (40 mL, 30%) gave a highly exothermic
reaction with an initial colour change from deep red to green and then to
brown. The brown solution was reduced in volume to approximately 10 mL,
passed through a Sephadex LH20 column and the brown fraction taken to
dryness (ca. 71% yield without attempted optimisation).
Fig. 1
‡ Microanalytical and other data are consistent with the formula HArg-
[Ru(Hedta)(NO)Cl]·H2O in which the cation is protonated arginine, present
in excess in the reaction medium. Found: C, 29.42; H, 4.64; N, 15.43.
HArg[Ru(Hedta)(NO)Cl]·H2O (C16H30N7O12ClRu) requires C, 29.61; H,
4.66; N, 15.11%.
Reaction of K[Ru(Hedta)Cl]3,6 with a four-fold excess of
-
arginine in the presence of hydrogen peroxide in aqueous
solution, gave in a highly exothermic reaction, a brown solution
which, following purification on a Sephadex LH 20 column and
L
1870
Chem. Commun., 2001, 1870–1871
This journal is © The Royal Society of Chemistry 2001
DOI: 10.1039/b106097j