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Reactions of molybdenum compounds in solution — model reactions for biological systems
Journal of the Less-Common
Metals, 36 (1974) 465 - 474 @ Elsevier Sequoia S.A., Lausanne - Printed in The Netherlands
REACTIONS MODEL
OF MOLYBDENUM
REACTIONS
FOR
COMPOUNDS
BIOLOGICAL
465
IN SOLUTION
-
SYSTEMS*
JACK T. SPENCE** and PETER KRONEKt Department
of Chemistry and Biochemistry,
Utah State University, Logan, Utah, 84322
(U.S.A.)
SUMMARY
Kinetic investigations of the reduction of flavins by MO(V) complexes in aqueous solution as model systems for Mo-flavin interaction in xanthine oxidase and other molybdenum enzymes are discussed. Data are presented indicating that MO(V)-dimers may react by either one- or two-electron mechanisms, depending on conditions and ligand environment. The reduction of flavin mononucleotide by the model complex, I.c-oxo-bis[oxodihydroxo(L-cysteinato)molybdate(V)] , is reported in detail, and mechanisms for its reaction under a variety of conditions are presented. The reduction of this complex with NaBH4 to produce a Mo(III) species is also discussed, and investigations of the oxidation of aldehydes by Mo(VI)-thiol complexes in non-aqueous solvents are reported.
INTRODUCTION
Currently, there is considerable interest in the chemistry of molybdenum. This is mainly due to the fact that molybdenum is an essential cofactor of the enzymes catalysing important reactions such as nitrate and nitrogen reduction as well as purine and aldehyde oxidation in a large variety of organisms [l] . Despite the obvious importance of these reactions, the nature of the active site and the function of the metal in the redox processes are not well understood. The results of the biochemical investigations for a number of molybdenum enzymes and the current status of model studies of the role of molybdenum in nitrogen reduction will be discussed by other participants in this conference. This paper will be concerned mainly with molybdenum-flavin reactions and model studies for purine oxidation.
* Presented at the Conference on “The Chemistry and Uses of Molybdenum”, at the University of Reading, England, September 17 - 21, 1973, sponsored by Climax Molybdenum Co. Ltd., and the Chemical Society (Dalton Division). **To whome correspondence should be addressed. t Present address: Fachbereich Biologie, Universitlt Konstanz, D-7750, Konstanz, Germany.
466 MOLYBDENUM-FLAVIN
REACTIONS
All of the molybdenum enzymes are extremely complex, containing flavin and heme or non-heme iron components in addition to molybdenum. With xanthine oxidase, the most extensively studied among these enzymes, the original rapid freeze e.s.r. results suggested the following pathway of electron transfer [l] Substrate + MO -+ FAD + Fe-S*
+ OZ.
More recently, a pooled system with molybdenum equivalents, has been proposed [ 1]
(1) as the entrance point for reducing
FAD + O2 / Substrate + Mo~P~~,*
For nitrate reductases, the electron transfer scheme appears to be NADPH -+ FAD + MO + Fe(cyt or Fe-S*) (ii) I (i)
+ NO;
(2)
(several nitrate reductases contain heme iron [ 1] , at least one has non-heme iron [2] , and some do not have iron at all [ 11). Clearly, model reactions between flavins and molybdenum complexes are of considerable interest with regard to the information they might give concerning molybdenum-flavin interaction in these enzymes. Preliminary studies of the reaction between flavin mononucleotide (FMN) and the Mo(V, VI) redox couple were reported by Mitchell and Williams [3] . They found that [MoOC15] 2- partially reduces FMN to FMNH2 in basic solution, whilst [MoOa] 2partially oxidises FMNHa under the same conditions. Additionally, increased visible absorption, attributed to flavo-semiquinone, was observed in partially reduced solutions containing [MoOa] 2: More detailed studies of the reactions between the Mo(V, VI) redox couple and flavins were reported by Hemmerich and Spence [4] and Spence et al. [S] . Using lumiflavin-3-acetic acid, they found that a pH-dependent equilibrium is established in phosphate buffer, pH 6 - 8 Flti
+ 2Mo(VI) f Fl,, + MO(V), .
(3)
(MO(V)? and Mo(V1) are most probably present as phosphate complexes, but their structures are unknown.) At pH 6.0, the reaction proceeds essentially completely to the right. As the pH is raised, the equilibrium shifts to the left, approaching completion in that direction at pH = 8.0. During the course of the forward reaction, a deep red, transient colour was observed, the spectrum of which suggested a metal-flavosemiquinone complex. The following structure was assigned to the e.s.r.-inactive complex.
(i) Nicotinamide adenine dinucleotide phosphate (NADPH). (ii) Flavine adenine dinucleotide (FAD).
467
It would therefore appear that the reaction proceeds in a one-electron step, although this was not unambiguously proven by a detailed kinetic investigation. Recently, a kinetic study of the reduction of FMN by MO(V) in tartrate buffer, pH = 2.5 - 5.0, was completed in this laboratory [6], Tartrate was chosen since other work has shown that the MO(V) monomer is somewhat stabilised in this buffer [7]. Again, a pH-dependent equilibrium was found, with high pH favouring reactants and low pH favouring products. Because of experimental considerations, the reverse reaction was used for kinetic studies, and its rate investigated over a wide range of conditions. Contrary to previous results, the kinetics of the reaction in tartrate are not compatible with a oneelectron transfer mechanism and could only be rationalised in terms of a two-electron step involving MO(W) as a reactive intermediate MO(V), P Mo(IV) + Mo(V1)
(4)
Mo(IV) -I-Fl,, P Mo(VI) f Fired.
(5)
Applying the steady-state
treatment
kg W(V)2 -d [ Fl,,] /dz = -
to MofIV) gives the rate-expression
I t&,x1
k-&s
-
NW)1 2 P%d I& I
[Mo(VI)I + [Floxl
Since the equilibrium constant, K,, could be determined experimentally, this expression can be integrated when both MO(V), and Mo(V1) are present in large excess. A computercurve-fitting-programme was used to find the best value of k-Jk5, which allowed both k4 and k5 to be evaluated from the plots, The integrated expression was found to fit the data to 90 - 95% of the reaction and to give consistent rate constants. No evidence was obtained for a red Me(V)-~avosemiquinone intermediate, a result expected for a twoelectron step rneG~ni~. Considerable evidence indicates that the site of molybdenum binding in xanthine oxidase and other enzymes is the mercapto group of a cysteine residue ]I 1. Consequently molybdenum sulphydryl complexes have been of great interest as models for the active site. Recently, we have been investigating the properties of the binuclear dioxobridged MO(V)-cysteine complex, di-p-oxo-bis[oxo(L-cysteinata) molybdate(V)] (1) the structure of which has been determined by X-ray crystallography 181. 2-
0 ~~
I I 0
0
N
ci ‘1~ MO 0 01
I 0
S 0
468
We have found that complex (1) undergoes bridge cleavage in basic solution (pH 8 - 11) to give the monoxobridged complex, p-oxo-bis[oxodihydroxo-(L-cysteinato) molybdate
091~(2) ]9,101. 0
0
s\Jo/o\?6”coo-
N’I\,,0l.P1
Hz0
(1)+20H-.2
-0oc
(6)
OH
HO
(2) Complex (2) in contrast to complex (1) was found to reduce FMN rapidly, thus serving as an interesting model for electron transfer between molybdenum and flavin in xanthine oxidase. Fl,, t (2) -+ 2 [Moo41 ‘- t 2 cysteine t Fir,+
(7)
Furthermore, solutions of (2) exhibit a broad absorption maximum near 600 nm, and a weak e.s.r. signal due to the MO(V) monomer in equilibrium with (2), both of which are quite similar to the corresponding spectra of xanthine oxidase [ 11, 121. It was found that the addition of cysteine increases the rate of reaction (7). Recent work by Gibian, et al., has indicated that a very slow reduction of FMN by cysteine does occur, but its rate is insignificant compared with the rate of reduction by complex (2) [13]. It was therefore concluded that added cysteine catalyses the reduction of FMN by (2). Both the catalysed and uncatalysed reactions were studied in the pH range 8 - 11 and were found to be first order in (2) and fist order in flavin. Both reactions contribute to the rate under these conditions, giving rise to the complete rate expression for uncatalysed and catalysed reactions: -d[Fl,,]
/dt = k,‘[Fl,,]
[(2)] t k7”[F1,,]
[(2)] [cysteine] .
The pH dependency of the rate constant for both reactions is complex (see Fig. 1). In order to explain these results, the acidic properties of the N-3 proton of the flavin nucleus and cysteine have to be taken into account.
0
0
%xH
pKa = 10.28
25 ‘C, /.L= 0.10
Fl,x-
R = CH2(CHOH)JCHzOF’Oa2Cysteine exists in five forms in solution, depending on pH [ 141. The most reasonable explanation for the catalytic effect of cysteine is the formation of an intermediate complex with FMN which is reduced rapidly by (2). Such a complex
469
IL 8.5cl
900
9.50
pH
I 0.m
Fig. I. pH dependence on observed rate constant, fog k,, for the catalyzed reaction of FMN and the MO(V)-cyst&e complex (2) in the presence of added cysteine. 0.0385 M cysteine, 0.50 BIborate, 25 “C. Full line, experimental; broken line, caEculated curve (see text). (Ref. 181
HS-CH&H-COOH
h
pK, 2-00
pK2 8.5.3 HS-CH,-CH-COO-’
I NHs+
.
“’S-CH&H-COO I NH,+
I NH,
Lf
NH, 4-
NH2 Lz-
might involve substitution of cysteine at position 4, or 5 of the flavin nucleus, in agreement with known properties af flavins [ 15) .
470 l? =
-CH2(CHOH)3CH20P032-
R’ = -CH,CH(NHa+)COO-
or -CHzCH(NHz)COO:
A similar complex has been proposed for the reduction of flavins by mercaptans [ 131. Assuming both FMN (Fl,,H) and its anion (FL,,-) react with (2) in the uncatalysed reaction, and intermediate complexes of the type discussed are formed between Li and L2- and both Fl,,H and Fl,,, which subsequently react in fast steps with (2) in the catalysed reaction, the following mechanism may be written Fl,,H Fl,,
t(2)
k8
p Products
(8)
t (2)
k9
l
Products
(9)
F&H k
t Lr-
10
Cl
(10)
kll ’
Products
(11)
k g’ k-1,
c2
(12)
KC,
-=
k-1, Cl t(2) Fl,,H
+ L2-
k 12 -
go k-1,
Kc,
=
k-1, c2 t(2)
k13>Products
(13)
Fl,,t L,,-
2’
(14)
c3
k-a
k 14
'Kc,
-
k-.
14
c3 t (2) Fl,,
+
k15Products
(15)
k
c4
(16)
Products
(17)
b
L2-
k-m k 16 -
=
k-1, C4 t (2)
Kc, k17
l
Applying the steady state treatment to complexes C r - Cq, assuming that the dissociation reactions of the complexes are much faster than their reactions with (2), and substituting in the rate expression from the appropriate equilibria for the proper forms of FMN and cysteine, gives the complete rate expression:
P%sl [WI -d [&J ldt = [H+l + K, kl ,&$,K,CL
lH+l
2 + kl
ks [H+] + k&
+
W’l + kl &,WG&CL
&$&&CL
[H’l + WQ,~&&&C~
[H’] 2K5 + [H+]K2(K4 + KS) + KzKaKs
~____.
(CL = total cysteine added; Fl,, = total oxidised FMN). At constant
pH and CL, this is identical with the experimental
-d [Fl,,l
ldt = k7
[FL,,1[WI
From this, the expression for the observed rate constant, kz([H+l +K,) - k,([H+J - k&J
k&c,&&G[H+12 At constant
+ kJG$dW&CL
CL, this is an equation
v=a[H+12
rate expression
k2, may be written
([H’l 2K~+ [H+l&(& +Ks) + K&I&) = W’l + bJGyWWW~
W’l + kdGy%LWWL.
of the form
+ b[H+] +c.
The data were processed by a computer programme to obtain the best values of a, b and c, and these were used to calculate the dashed curve of Fig. 1, giving good agreement with experiment over the pH range used. Using a flow system connected to a four jet mixing chamber, e.s.r. spectra of the reaction mixture were obtained at room temperature and 77 K. In both cases, MO(V) monomer (g 1.974) and flavosemiquinone radical (g 2.003) signals were obtained, whilst solutions of (2) or flavin alone gave no signal under these conditions (Fig. 2). The flavin radical concentration was approximately equivalent to that observed with solutions containing .the same amounts of oxidised and reduced FMN. The MO(V) monomer signal has the sameg-value and hyperfine splitting reported earlier for the MO(V)-cysteine complex [ 121 . The e.s.r. and kinetic results clearly indicate that the reduction proceeds, at least partially, by a one-electron step. The MO(V) e.s.r. signal must arise from the removal of one electron from (2), forming a MO(V) monomer and a flavosemiquinone @lHJS. The monomer may then either dimerise to (2) or react with a second FMN, giving products. The flavosemiquinone radical disproportionates to give oxidised and reduced FMN, a reaction known to be extremely rapid [ 161. Fl,, t (2) -P [Moo41 *- t [MoO(OH)2(cysteine)]
- + l$H-
2 i;lH- P Fl,, t Fl,,dH2 [MoO(OH), (cysteine)]
(18) (19)
- + (2)
Fl,, t [M~O(OH)~(cysteine)]
(20)
- --f [Moos] *- + cysteine t filH:
$ In the pH range used, flavosemiquinone
exists as FIH:
(21)
412
Fig. 2. E.s.r. spectra of F&,x- plus Mo(Vf-cysteine complex (2) reaction mixture (continuous flow); 0.040 M cysteine, 5.0 X low3 MFl,x, 1.25 X lo-” M (2), pH 10.00, OSOMborate, 25 “C. (Ref. 18.)
Applying the steady state treatment to the flavosemiquinone radical and the MO(V) monomer, and assuming that reactions (19), (20) and (21) are fast with respect to (18), leads to a rate expression identical with the experimental expression -d W,,l Idt = k~s Woxl WI . Similar treatment for the catalysed reaction also gives the same experimental expression. Studies of xanthine oxidase have indicated that the substrate may reduce the molybdenum component not only to the (V) state, but also to a lower state, possibly (IV). The results of the model reactions discussed here indicate that flavin can react with reduced molybdenum by both one and two electron steps. In the presence of the substrate, e.s.r. signals for both MO(V) and flavosemiquinone are observed with xanthine oxidase [l] . Although these results have been interpreted in terms of two monomeric non-interacting MO(V) sites in the enzyme [l] , the data reported here show that substantial MO(V) e.s.r. signals, without evidence of metal-metal interaction, can arise from one-electron oxidation of spin-coupled Mo(V)-dimers. Therefore, the presence of such dimeric structures in the enzymes should not be ruled out. 7 CH,
I+H-
473
Reduction of Mo( V)-cysteine complexes by NaBH, Recent work on the reduction of xanthine oxidase by substrate has suggested that the molybdenum may be reduced beyond the (V) level to a (IV) or a (III) state. Similarly, Schrauzer et al. [ 171, in their model studies on nitrogenase, proposed a Mo(IV)-cysteine complex as the active catalyst. Consequently, we have initiated an investigation of the reduction of MO(V)-cysteine complexes (1) and (2) by NaBHa. Preliminary results indicate that the reduction proceeds by bridge cleavage, producing a high level of a monomeric MO(V) intermediate, as detected by quantitative e.s.r. spectrometry. Eventually, the MO(V)-complex is reduced to an Mo(II1) species, as determined by comparison with electronic spectra of known Mo(II1) compounds and magnetic susceptibility measurements of the product. BH; (1) + -
2 [MoO(OH)z(cysteine)]
-
BH,-
[MoO(OH)2(cysteine)]
- B
Mo(III)-cysteine.
The structure of the Mo(III) species has not yet been determined, but it is probably cysteine complex. Work on the detailed mechanism of this reaction is in progress.
a
Oxidation of aldehydes by Mo( VI) complexes In basic solution (pH 8 - 10) few, if any, Mo(V1) complexes are obtainable, due to the great stability of the [MOO,] ‘- ion. In non-aqueous solvents, however, Mo(V1) complexes may be easily prepared. As a model for the first step in the oxidation of the substrate by xanthine oxidase we studied the oxidation of aldehydes by Mo(VI)-thiol complexes in DMF and DMSO. Initial results indicate that aldehydes are oxidised by the Mo(VI)-cysteine ethyl ester complex (3) to give the corresponding acid, with the production of a binuclear MO(V)-complex
RCHO + Mo02L,
0 I ,o,? j L2Mo
MoL2 + RCOOH
(L = -S-CH2-CH(NH2)-COOCH2CH3). This is formally equivalent to an oxygen atom transfer from the Mo(VI)-complex to the substrate and may represent a general mechanism for such oxidations. Work on the kinetics and mechanism of this reaction is continuing. CONCLUSIONS
Kinetic studies of the reduction of flavins by MO(V)-complexes under a variety of conditions have shown that the reaction may proceed by both one or two electron steps, depending primarily on the ligands. These results support current hypotheses of electron transfer between molybdenum and flavin cofactors of xanthine oxidase in which both MO(V) and Mo(IV) species are proposed to reduce the flavin. E.s.r. studies also show the presence of intermediate MO(V) monomers during redox reactions of e.s.r.-inactive spincoupled MO(V) complexes. These experimental results provide a possible explanation for the appearance of large MO(V) e.s.r. signals during reactions catalysed by molybdenum
474
enzymes. Finally, ho-thiol complexes will oxidise aidehydes in solvents such as DMF or DMSO. This suggests that the Mo(V1) of xanthine oxidase is located in a nonaqueous environment, thus preventing hydrolysis to the highly stable [MOO,] 2- ion. REFERENCES 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18
R. C. Bray and J. C. Swann, Structure and Bonding, 11 (1972) 107. P. Forget and D. V. Devartanian, Biochim. Biophys. Acta, 256 (1972) 600. P. C. H. Mitchell and R. J. P. Williams, Biochim. Biophys. Acta, 86 (1964) 39. P. Hemmerich and J. T. Spence, in E. C. Slater ted.), Flavins and Flavoproteins, Elsevier, Amsterdam, 1966, p. 82. J. T. Spence, M. Heydanek and P. Hemmerich, in A. Ehrenberg, B. G. M~mstr~m and T. VInng&d (eds.), Magnetic Resonance in Biological Systems, Pergamon, Oxford, 1967, p. 269. G. Colovos and J. T. Spence, Biochemistry, 11 (1972) 2452. J. T. Spence and M. Heydanek, Inorg. Chem., 6 (1967) 1489. J. R. Knox and C. J. Prout, ActaCryst., B25 (1969) 1857. P. Kroneck and J. T. Spence, Inorg. Nucl. Chem. Letters, 9 (1973) 177. P. Kroneck and J. T. Spence, J. Inorg. Nucl. Chem., 3.5 (1973) 3391. K. Garbett, R. D. Gillard, P. F. Knowles and J. E. Stangroom, Nature, 215 (1967) 824. T. J. Huang and G. P. Haight, Jr., J. Am. Chem. Sot., 92 (1970) 2336. M. .I. Gibian, D. L. Elliot, C. Kelley, B. Borge and K. Kupecz, Z. Naturforsch., 27b (1972) 1016. R. E. Benesch and R. Benesch, J. Am. Chem. Sot., 77 (195.5) 5877. L. E. Brown and G. H. Hamilton, J. Am. Chem. Sot., 92 (1970) 7225. G. B. Barman and G. Tollin, Biochemistry, I1 (1972) 4760. G. N. Schrauzer and P. A. Doemeny, J. Am. Chem. Sot., 93 (1971) 1608. J. T. Spence and P. Kroneck, Biochemistry, 12 (1973) 5050.
Metals, 36 (1974) 465 - 474 @ Elsevier Sequoia S.A., Lausanne - Printed in The Netherlands
REACTIONS MODEL
OF MOLYBDENUM
REACTIONS
FOR
COMPOUNDS
BIOLOGICAL
465
IN SOLUTION
-
SYSTEMS*
JACK T. SPENCE** and PETER KRONEKt Department
of Chemistry and Biochemistry,
Utah State University, Logan, Utah, 84322
(U.S.A.)
SUMMARY
Kinetic investigations of the reduction of flavins by MO(V) complexes in aqueous solution as model systems for Mo-flavin interaction in xanthine oxidase and other molybdenum enzymes are discussed. Data are presented indicating that MO(V)-dimers may react by either one- or two-electron mechanisms, depending on conditions and ligand environment. The reduction of flavin mononucleotide by the model complex, I.c-oxo-bis[oxodihydroxo(L-cysteinato)molybdate(V)] , is reported in detail, and mechanisms for its reaction under a variety of conditions are presented. The reduction of this complex with NaBH4 to produce a Mo(III) species is also discussed, and investigations of the oxidation of aldehydes by Mo(VI)-thiol complexes in non-aqueous solvents are reported.
INTRODUCTION
Currently, there is considerable interest in the chemistry of molybdenum. This is mainly due to the fact that molybdenum is an essential cofactor of the enzymes catalysing important reactions such as nitrate and nitrogen reduction as well as purine and aldehyde oxidation in a large variety of organisms [l] . Despite the obvious importance of these reactions, the nature of the active site and the function of the metal in the redox processes are not well understood. The results of the biochemical investigations for a number of molybdenum enzymes and the current status of model studies of the role of molybdenum in nitrogen reduction will be discussed by other participants in this conference. This paper will be concerned mainly with molybdenum-flavin reactions and model studies for purine oxidation.
* Presented at the Conference on “The Chemistry and Uses of Molybdenum”, at the University of Reading, England, September 17 - 21, 1973, sponsored by Climax Molybdenum Co. Ltd., and the Chemical Society (Dalton Division). **To whome correspondence should be addressed. t Present address: Fachbereich Biologie, Universitlt Konstanz, D-7750, Konstanz, Germany.
466 MOLYBDENUM-FLAVIN
REACTIONS
All of the molybdenum enzymes are extremely complex, containing flavin and heme or non-heme iron components in addition to molybdenum. With xanthine oxidase, the most extensively studied among these enzymes, the original rapid freeze e.s.r. results suggested the following pathway of electron transfer [l] Substrate + MO -+ FAD + Fe-S*
+ OZ.
More recently, a pooled system with molybdenum equivalents, has been proposed [ 1]
(1) as the entrance point for reducing
FAD + O2 / Substrate + Mo~P~~,*
For nitrate reductases, the electron transfer scheme appears to be NADPH -+ FAD + MO + Fe(cyt or Fe-S*) (ii) I (i)
+ NO;
(2)
(several nitrate reductases contain heme iron [ 1] , at least one has non-heme iron [2] , and some do not have iron at all [ 11). Clearly, model reactions between flavins and molybdenum complexes are of considerable interest with regard to the information they might give concerning molybdenum-flavin interaction in these enzymes. Preliminary studies of the reaction between flavin mononucleotide (FMN) and the Mo(V, VI) redox couple were reported by Mitchell and Williams [3] . They found that [MoOC15] 2- partially reduces FMN to FMNH2 in basic solution, whilst [MoOa] 2partially oxidises FMNHa under the same conditions. Additionally, increased visible absorption, attributed to flavo-semiquinone, was observed in partially reduced solutions containing [MoOa] 2: More detailed studies of the reactions between the Mo(V, VI) redox couple and flavins were reported by Hemmerich and Spence [4] and Spence et al. [S] . Using lumiflavin-3-acetic acid, they found that a pH-dependent equilibrium is established in phosphate buffer, pH 6 - 8 Flti
+ 2Mo(VI) f Fl,, + MO(V), .
(3)
(MO(V)? and Mo(V1) are most probably present as phosphate complexes, but their structures are unknown.) At pH 6.0, the reaction proceeds essentially completely to the right. As the pH is raised, the equilibrium shifts to the left, approaching completion in that direction at pH = 8.0. During the course of the forward reaction, a deep red, transient colour was observed, the spectrum of which suggested a metal-flavosemiquinone complex. The following structure was assigned to the e.s.r.-inactive complex.
(i) Nicotinamide adenine dinucleotide phosphate (NADPH). (ii) Flavine adenine dinucleotide (FAD).
467
It would therefore appear that the reaction proceeds in a one-electron step, although this was not unambiguously proven by a detailed kinetic investigation. Recently, a kinetic study of the reduction of FMN by MO(V) in tartrate buffer, pH = 2.5 - 5.0, was completed in this laboratory [6], Tartrate was chosen since other work has shown that the MO(V) monomer is somewhat stabilised in this buffer [7]. Again, a pH-dependent equilibrium was found, with high pH favouring reactants and low pH favouring products. Because of experimental considerations, the reverse reaction was used for kinetic studies, and its rate investigated over a wide range of conditions. Contrary to previous results, the kinetics of the reaction in tartrate are not compatible with a oneelectron transfer mechanism and could only be rationalised in terms of a two-electron step involving MO(W) as a reactive intermediate MO(V), P Mo(IV) + Mo(V1)
(4)
Mo(IV) -I-Fl,, P Mo(VI) f Fired.
(5)
Applying the steady-state
treatment
kg W(V)2 -d [ Fl,,] /dz = -
to MofIV) gives the rate-expression
I t&,x1
k-&s
-
NW)1 2 P%d I& I
[Mo(VI)I + [Floxl
Since the equilibrium constant, K,, could be determined experimentally, this expression can be integrated when both MO(V), and Mo(V1) are present in large excess. A computercurve-fitting-programme was used to find the best value of k-Jk5, which allowed both k4 and k5 to be evaluated from the plots, The integrated expression was found to fit the data to 90 - 95% of the reaction and to give consistent rate constants. No evidence was obtained for a red Me(V)-~avosemiquinone intermediate, a result expected for a twoelectron step rneG~ni~. Considerable evidence indicates that the site of molybdenum binding in xanthine oxidase and other enzymes is the mercapto group of a cysteine residue ]I 1. Consequently molybdenum sulphydryl complexes have been of great interest as models for the active site. Recently, we have been investigating the properties of the binuclear dioxobridged MO(V)-cysteine complex, di-p-oxo-bis[oxo(L-cysteinata) molybdate(V)] (1) the structure of which has been determined by X-ray crystallography 181. 2-
0 ~~
I I 0
0
N
ci ‘1~ MO 0 01
I 0
S 0
468
We have found that complex (1) undergoes bridge cleavage in basic solution (pH 8 - 11) to give the monoxobridged complex, p-oxo-bis[oxodihydroxo-(L-cysteinato) molybdate
091~(2) ]9,101. 0
0
s\Jo/o\?6”coo-
N’I\,,0l.P1
Hz0
(1)+20H-.2
-0oc
(6)
OH
HO
(2) Complex (2) in contrast to complex (1) was found to reduce FMN rapidly, thus serving as an interesting model for electron transfer between molybdenum and flavin in xanthine oxidase. Fl,, t (2) -+ 2 [Moo41 ‘- t 2 cysteine t Fir,+
(7)
Furthermore, solutions of (2) exhibit a broad absorption maximum near 600 nm, and a weak e.s.r. signal due to the MO(V) monomer in equilibrium with (2), both of which are quite similar to the corresponding spectra of xanthine oxidase [ 11, 121. It was found that the addition of cysteine increases the rate of reaction (7). Recent work by Gibian, et al., has indicated that a very slow reduction of FMN by cysteine does occur, but its rate is insignificant compared with the rate of reduction by complex (2) [13]. It was therefore concluded that added cysteine catalyses the reduction of FMN by (2). Both the catalysed and uncatalysed reactions were studied in the pH range 8 - 11 and were found to be first order in (2) and fist order in flavin. Both reactions contribute to the rate under these conditions, giving rise to the complete rate expression for uncatalysed and catalysed reactions: -d[Fl,,]
/dt = k,‘[Fl,,]
[(2)] t k7”[F1,,]
[(2)] [cysteine] .
The pH dependency of the rate constant for both reactions is complex (see Fig. 1). In order to explain these results, the acidic properties of the N-3 proton of the flavin nucleus and cysteine have to be taken into account.
0
0
%xH
pKa = 10.28
25 ‘C, /.L= 0.10
Fl,x-
R = CH2(CHOH)JCHzOF’Oa2Cysteine exists in five forms in solution, depending on pH [ 141. The most reasonable explanation for the catalytic effect of cysteine is the formation of an intermediate complex with FMN which is reduced rapidly by (2). Such a complex
469
IL 8.5cl
900
9.50
pH
I 0.m
Fig. I. pH dependence on observed rate constant, fog k,, for the catalyzed reaction of FMN and the MO(V)-cyst&e complex (2) in the presence of added cysteine. 0.0385 M cysteine, 0.50 BIborate, 25 “C. Full line, experimental; broken line, caEculated curve (see text). (Ref. 181
HS-CH&H-COOH
h
pK, 2-00
pK2 8.5.3 HS-CH,-CH-COO-’
I NHs+
.
“’S-CH&H-COO I NH,+
I NH,
Lf
NH, 4-
NH2 Lz-
might involve substitution of cysteine at position 4, or 5 of the flavin nucleus, in agreement with known properties af flavins [ 15) .
470 l? =
-CH2(CHOH)3CH20P032-
R’ = -CH,CH(NHa+)COO-
or -CHzCH(NHz)COO:
A similar complex has been proposed for the reduction of flavins by mercaptans [ 131. Assuming both FMN (Fl,,H) and its anion (FL,,-) react with (2) in the uncatalysed reaction, and intermediate complexes of the type discussed are formed between Li and L2- and both Fl,,H and Fl,,, which subsequently react in fast steps with (2) in the catalysed reaction, the following mechanism may be written Fl,,H Fl,,
t(2)
k8
p Products
(8)
t (2)
k9
l
Products
(9)
F&H k
t Lr-
10
Cl
(10)
kll ’
Products
(11)
k g’ k-1,
c2
(12)
KC,
-=
k-1, Cl t(2) Fl,,H
+ L2-
k 12 -
go k-1,
Kc,
=
k-1, c2 t(2)
k13>Products
(13)
Fl,,t L,,-
2’
(14)
c3
k-a
k 14
'Kc,
-
k-.
14
c3 t (2) Fl,,
+
k15Products
(15)
k
c4
(16)
Products
(17)
b
L2-
k-m k 16 -
=
k-1, C4 t (2)
Kc, k17
l
Applying the steady state treatment to complexes C r - Cq, assuming that the dissociation reactions of the complexes are much faster than their reactions with (2), and substituting in the rate expression from the appropriate equilibria for the proper forms of FMN and cysteine, gives the complete rate expression:
P%sl [WI -d [&J ldt = [H+l + K, kl ,&$,K,CL
lH+l
2 + kl
ks [H+] + k&
+
W’l + kl &,WG&CL
&$&&CL
[H’l + WQ,~&&&C~
[H’] 2K5 + [H+]K2(K4 + KS) + KzKaKs
~____.
(CL = total cysteine added; Fl,, = total oxidised FMN). At constant
pH and CL, this is identical with the experimental
-d [Fl,,l
ldt = k7
[FL,,1[WI
From this, the expression for the observed rate constant, kz([H+l +K,) - k,([H+J - k&J
k&c,&&G[H+12 At constant
+ kJG$dW&CL
CL, this is an equation
v=a[H+12
rate expression
k2, may be written
([H’l 2K~+ [H+l&(& +Ks) + K&I&) = W’l + bJGyWWW~
W’l + kdGy%LWWL.
of the form
+ b[H+] +c.
The data were processed by a computer programme to obtain the best values of a, b and c, and these were used to calculate the dashed curve of Fig. 1, giving good agreement with experiment over the pH range used. Using a flow system connected to a four jet mixing chamber, e.s.r. spectra of the reaction mixture were obtained at room temperature and 77 K. In both cases, MO(V) monomer (g 1.974) and flavosemiquinone radical (g 2.003) signals were obtained, whilst solutions of (2) or flavin alone gave no signal under these conditions (Fig. 2). The flavin radical concentration was approximately equivalent to that observed with solutions containing .the same amounts of oxidised and reduced FMN. The MO(V) monomer signal has the sameg-value and hyperfine splitting reported earlier for the MO(V)-cysteine complex [ 121 . The e.s.r. and kinetic results clearly indicate that the reduction proceeds, at least partially, by a one-electron step. The MO(V) e.s.r. signal must arise from the removal of one electron from (2), forming a MO(V) monomer and a flavosemiquinone @lHJS. The monomer may then either dimerise to (2) or react with a second FMN, giving products. The flavosemiquinone radical disproportionates to give oxidised and reduced FMN, a reaction known to be extremely rapid [ 161. Fl,, t (2) -P [Moo41 *- t [MoO(OH)2(cysteine)]
- + l$H-
2 i;lH- P Fl,, t Fl,,dH2 [MoO(OH), (cysteine)]
(18) (19)
- + (2)
Fl,, t [M~O(OH)~(cysteine)]
(20)
- --f [Moos] *- + cysteine t filH:
$ In the pH range used, flavosemiquinone
exists as FIH:
(21)
412
Fig. 2. E.s.r. spectra of F&,x- plus Mo(Vf-cysteine complex (2) reaction mixture (continuous flow); 0.040 M cysteine, 5.0 X low3 MFl,x, 1.25 X lo-” M (2), pH 10.00, OSOMborate, 25 “C. (Ref. 18.)
Applying the steady state treatment to the flavosemiquinone radical and the MO(V) monomer, and assuming that reactions (19), (20) and (21) are fast with respect to (18), leads to a rate expression identical with the experimental expression -d W,,l Idt = k~s Woxl WI . Similar treatment for the catalysed reaction also gives the same experimental expression. Studies of xanthine oxidase have indicated that the substrate may reduce the molybdenum component not only to the (V) state, but also to a lower state, possibly (IV). The results of the model reactions discussed here indicate that flavin can react with reduced molybdenum by both one and two electron steps. In the presence of the substrate, e.s.r. signals for both MO(V) and flavosemiquinone are observed with xanthine oxidase [l] . Although these results have been interpreted in terms of two monomeric non-interacting MO(V) sites in the enzyme [l] , the data reported here show that substantial MO(V) e.s.r. signals, without evidence of metal-metal interaction, can arise from one-electron oxidation of spin-coupled Mo(V)-dimers. Therefore, the presence of such dimeric structures in the enzymes should not be ruled out. 7 CH,
I+H-
473
Reduction of Mo( V)-cysteine complexes by NaBH, Recent work on the reduction of xanthine oxidase by substrate has suggested that the molybdenum may be reduced beyond the (V) level to a (IV) or a (III) state. Similarly, Schrauzer et al. [ 171, in their model studies on nitrogenase, proposed a Mo(IV)-cysteine complex as the active catalyst. Consequently, we have initiated an investigation of the reduction of MO(V)-cysteine complexes (1) and (2) by NaBHa. Preliminary results indicate that the reduction proceeds by bridge cleavage, producing a high level of a monomeric MO(V) intermediate, as detected by quantitative e.s.r. spectrometry. Eventually, the MO(V)-complex is reduced to an Mo(II1) species, as determined by comparison with electronic spectra of known Mo(II1) compounds and magnetic susceptibility measurements of the product. BH; (1) + -
2 [MoO(OH)z(cysteine)]
-
BH,-
[MoO(OH)2(cysteine)]
- B
Mo(III)-cysteine.
The structure of the Mo(III) species has not yet been determined, but it is probably cysteine complex. Work on the detailed mechanism of this reaction is in progress.
a
Oxidation of aldehydes by Mo( VI) complexes In basic solution (pH 8 - 10) few, if any, Mo(V1) complexes are obtainable, due to the great stability of the [MOO,] ‘- ion. In non-aqueous solvents, however, Mo(V1) complexes may be easily prepared. As a model for the first step in the oxidation of the substrate by xanthine oxidase we studied the oxidation of aldehydes by Mo(VI)-thiol complexes in DMF and DMSO. Initial results indicate that aldehydes are oxidised by the Mo(VI)-cysteine ethyl ester complex (3) to give the corresponding acid, with the production of a binuclear MO(V)-complex
RCHO + Mo02L,
0 I ,o,? j L2Mo
MoL2 + RCOOH
(L = -S-CH2-CH(NH2)-COOCH2CH3). This is formally equivalent to an oxygen atom transfer from the Mo(VI)-complex to the substrate and may represent a general mechanism for such oxidations. Work on the kinetics and mechanism of this reaction is continuing. CONCLUSIONS
Kinetic studies of the reduction of flavins by MO(V)-complexes under a variety of conditions have shown that the reaction may proceed by both one or two electron steps, depending primarily on the ligands. These results support current hypotheses of electron transfer between molybdenum and flavin cofactors of xanthine oxidase in which both MO(V) and Mo(IV) species are proposed to reduce the flavin. E.s.r. studies also show the presence of intermediate MO(V) monomers during redox reactions of e.s.r.-inactive spincoupled MO(V) complexes. These experimental results provide a possible explanation for the appearance of large MO(V) e.s.r. signals during reactions catalysed by molybdenum
474
enzymes. Finally, ho-thiol complexes will oxidise aidehydes in solvents such as DMF or DMSO. This suggests that the Mo(V1) of xanthine oxidase is located in a nonaqueous environment, thus preventing hydrolysis to the highly stable [MOO,] 2- ion. REFERENCES 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18
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