Revi!ta Mexicana de Fúica 38, Suplemento 1 (1992) 152-159

Eleetroehemieal studies on sorne Cu-Mo interaetiollS. I.New trends in bio-inorganic rnolybdenurn ehemistry*

ANTONIO QUIROZ-GUTIÉRREZ

Instituto de Física, Universidad Nacional Autónoma de MéxicoApartado postal 20-364, 01000 México, D.F., México

CECILIA JUÁREz.-GORDIANOInstituto de Investigaciones Biomédicas

Universidad Nacional Autónoma de MéxicoApartado postal 70-228, 04510 México, D.F., México

TOMÁS HERNÁNDEZ P. AND IGNACIO GONZÁLEZDepartamento de Química

Universidad Autónoma Metropolitana-IztapalapaApartado postal 55-534, Og340 México, D.F., México

Recibido el 3 de septiembre de 1991; aceptado el 31 de enero de 1992

ABSTRACT. Although molybdenum is an indispensable element for life since the Mc>-containingenzyrnes of the nitrogen cycle are crucial for nitrogen fixation and nitrate rcdudion, the copper-rnolybdenurn interactions in biological systerns rnake this elernent even more important since itcan form Cu-Mo complexes biologically inactives and it is able to modify the Cu biochemistry. Cumetalloproteins frequently display unusual coordination properties which are difficult to explainon the basis of inorganic chemistry. They are imparted by the protein dynamics that create specialgeornetrical environments strongly ¡n"uenccd by vibronic interactions as the Jahn- Teller effect,studied by the solid state physicists. Mo can then be used to improve our understanding of Cubehavior in biological systems. This physicc>-chemical knowledge will be specially important forthe biornedical sciences, since the Cu-Mo complexes behave as biologically inactives, and theseinteractions can be related with the oxidation states of both transition rnctals. Mo can be the keyto manipulate the Cu atoms in Wilsson, Menke, autoirnmmune diseases as rheumatoid arthritis,cancer and acquired immunodefficiency syndrome. We present in this colloquium our first report ona modified amoniurn tetrathiomolydate synthetized in Mexico, its electrochemica1 characterizationusing the Cu ions in the form of IIistidyl-hist-Cu and his-Cu complexes, with the idea of utilizingditrerent oxidation states of Mo to rernove other particular oxidation and rnagnetic states of Cu.

RESUMEN. Si bien el Mo es un elemento indispensable para la vida, y las molibdc>-enzimas del ciclodel N son cruciales en la fijación del N y la reducción del nitrato, sus interacciones con el Cu enlos seres vivos, hacen a este elemento aun más importante, ya que forma complejos Mo-Cu capacesde modificar la bioquímica del eu. Esta ha sido difícil de estudiar ya que las cupro-proteínasmuestran propiedades de coordinación insólitas y difíciles de explicar, debidas a la dinámica dela proteína, que aunada a los efectos vibracionales del metal, como el efecto Jahn-Teller, creanarreglos geométricos especiales, estudiados por la física del estado sólido. El Mo puede ser utilizadopara mejorar nuestro conocimiento del comportamiento del Cu en biología y este conocimiento,

*Supported by SETECOMFIA-UAM-Azcapotzalco.

ELECTHOCIIEMICAL STUOIES ON SOME Cu-Mo INTERACTIONS. I... 153

rela.cionado con la inl(>racción de los múltiples estados de oxidación y de valencia de ambos metalesde transición, será de especial importancia en biomedicina, ya que los complejos Mo-Cu, que secomportan corno 1Jialógicamente inactivos, pueden extraer selectivamente al Cu en determinadosestados de oxidación. Los compuestos de Mo pueden ser la clave para manipular los átomos de Cuen las enfermedades de \Vilson y Menkc, en el daño autoinmunc, en cáncer y SIDA. Presentamosen este coloquio los primeros reportes de un tetratiomolibdalo modificado, sintetizado en México,su caracterización electroquímica y algunas de sus interacciones con los iones de CUt utilizandoéstos en la forma de complejos Histidil.lIislidina-Cu e His-Cu, con la idea de utilizar diferentesestados de oxidación del !\.lo para extraer otros estados de oxidación del eu.

PACS: 87.22.Fy

l. INTTlODIJCTION

Although molyhdenulII is one of the less avaiJable elemer.t.s of both the earth's crust anrithe s¡'a water, beinl( present in ~ JO-" % [1,2], it is an indispen8able elcment for Jife ontIJe ('arth, since molyhdetlulTI containiug euzyrnes are essenlial lo the nitrogen cycle asthey are in"ul""d in nitrogen fixation and in the reduction of nitrate. Molybdenllm has auniqllc role A.1lI01lg l.ransitioll IIlctals due lo its ehemical v8rsatBity ano complex properti('sthat favor hlOlogical funet iOlls. SOIflC relcvant properties include:

!. ~1()ly¡'d,'uulII has the ahility of forllling compounds with ,nost inorganic and organkliga"ds.

2. It has oxidation stales ran¡:;ing fl'Orn -11 to VI.

3. It is ahle to fOl'ln eoordination numhers from .1 ta 8.

.l.' I\lolybd('nulll forllls high coordinal.ion number compl~xes, espccially with sulphur [!j .

.j. 11.can be in low-potelltial 1II111tiredoxstates (Mo(VI) _ Mo(lII)) which can be sta-bilized in a'l"cous medilllll by ligands sllch as nitrogen, sulphur 01' oxygen dúnorsgenerally found in living systems [3).

6. It has the capacity 1.0 act in ato m transfer, which in conjunction with point 5, makesit a good relay for converting reducing power to atom transfer [41.

7. l\lolybdenllm is able to form oxo-complexes, ego 1\10= O.

8. It can form anionic hetel'Opolymolibdates, with very high molecular weights (up to3000) for inorganic electrolytes. This macroanionic property accollnts for its biologicalacl.ion as for example in controlling estrogen and androgen receptors, embryogenesisamI normal and malignant growth [5].

9. It can make complexes with copper (with 01' without sulphur). The particular bio-chemical behavior of this kind of complexes, makes Mo able to extract copper fromproteins without hiochemical damage even in vertebrates [6,7,8,9).

For this reasons molybdenum is one of the metals of choice for enzymes which catalyseoxidation-redllction pl'Ocesses and transfer 01'storage of elect.rons [31, and may be a naturalmechanism in controlJing copper hiochemistry.

154 ANTONIO QUIROZ-GUTIÉRREZ ET AL.

2. THE Cu-Mo INTERACTIONS IN BIOLOGICAL SYSTEMS

Though molybdenum is already important by its role in the nitrogen cycle, there arenew trends in its bio-inorganic chemistry which make it even more importan!. Thecopper-molybdennm interactions in biological systems will make this clement the keyfor improving our understanding of the copper behavior in living systems.The existence of a biological antagonism between copper and molybdenum is well known

since the forties, when it was demonstrated that high dietary molybdenum produces acopper deficiency in cattle that grazed forages containing high leveIs of molybdenum, andthat copper deficiency can be alleviated by supplying additiooal copper [IO,IIJ.Since molybdenum toxicity can be diminished by copper supplemeotation not only in

cattle but in sheep, rabbits, laboratory rats and chicks, it was snggested that the formationof Cu-Mo complexes renders the copper biologically inactive. The observation that CnSO.and Na. MoO. form a complex which precipitates in a near oeutral solution was usedto develop the hypothesis that copper bound in molybdenum complexes is biologicallyinactive [6,7].More recently the interaction Cu-Mo has been stndied in maize. In this plant he molyb-

oennm deficiency a!fects copper absorption, and copper deficiency at normal molybdennmlevels produce a decrease of the dry weight, chlorophyll levels and protein nitrogeo,bnt the activities of the cytochrome oxidase and nitrate reductase beco me more pro-nonnced 1121.

3. THE IMPORTANCE OF Cu BIOCHEMISTRY IN HUMAN AND YETERINARY MEDICINE

Copper biochemistry beca me a malter of interest mainly due to the relationships existingbetween several human aod veterinary diseases. Wilson disea.';e, charaderizeo by copperaccumulation, was known from the fifties and controlled by using chelation agents suchas penicilamide. Menke disease, a new familial illne", characterized by rapidly progressivecerebral degeneration, has been controlled by copper administration mainly in the formof L-His2-Cu complexoCopper is important in arthritis and rheumatic diseases since this metal is in the active

site of enzymes as the super-oxyde oismutase that eliminate the free radicals useel bythe immnne system during the inflammatory reaction 18]. Unfortnnately there are othersituations in human pathology like cancer io which it is not clear if the increased copperlevels present in the normal and malignant tissues of the patients, represent an homeostaticmechar.ism or a causal agent. Or eveo both situation exist due to differeot kiod of copperchemical rearrangements. The health of an experimeotal animal with induced cancer areimproved with diminutioo of tumoral activity by treatmeot with copper chelation agl'nts,bul the :same animal improvc evcn mor(' ir t hcy receivc an ext.ra dm:;p of ropppr plus the

copper chelators 18].These fiodlngs show lhe preseoce of more than ooe type of copper atom from the

physiological poiot of view, since they are io di!ferent oxidation states that give di!ferentchemical behavior to the proteios io which they are located. Ooe important goal io cancerbiology is a better understaoding of the relationships between malignant traosíormation,

ELECTROCIIEMICAL STUDlES ON SOME Cu-Mo INTERACTIONS. I... 155

progression and host defense mechanism, and the role of Cu ions and their differentoxidation and magnetic states during those different steps of malignancy.¡¡¡gh levels of copper have been found in both normal and malignant tissues of pa-

tients with cancer and different types of malignant tumors. A close relationship has beendetected between copper levels and tumoral activity in lymphomas and Hodgkin's dis-ease [13,14,1.5,16]; in acute lymphocytic leukemia [171, in broncongenic carcinoma [16,181,in breast cancer [19], in cancers of the digestive system [20], in melanoma [21], and os-teogenic sarcoma [22]. Abnormally high levels of copper have been found in cervix utericancer and his pre-cancerous lesions [23,241. In brain tumors a close relationship has beenestablished between malignancy and copper levels [25,26).Preliminary results in the treatment of patients with the acquired immunodeficiency

syndrome (AlDS) with penicilamine [27], the powerful copper chelation agent used forthe treatment of Wilson's disease, have been reported but it still is not known which arethe copper levels in the different tissues of AIDS patients.

4. COPPER METALLOPROTl;INS BEIIAVE AS UNIQUE COORDlNATION COMPOUND

Copper proteins frequently display unusual coordination properties which are difficult toexplain based on the knowledge of simpler small inorganic complexes generally synthetizedby inorganic chemists [28]. These unique chemical properties of the copper ions, that canbe observed in different kinds of vibrational spectra, are imparted by the protein to whichthe metal atoms are tightly bOllnd [29].Protein-Cu interactions are related to protein dy~amics and the electronic configuration

of copper complexes. Sinee the rt' configuration of Cu2+ involves a single electron hole,their electron cloud is not spherically symmetrical, so that Cu2+ ligands generally forman irregular, elongated octahedron. Since ligands close to half-empty orbital are attractedowiug to the smaller shielding from the positive nucleus as compared with the ligandsclase to filled orbitals, s1leh particular geometric arrangements are generally referred toas the J"hn-Teller elreel [29].Cu2+ forms the strongest complexes with most ligands and is found in three main

forms in proteins: two paramagnetic forms normally designated as type 1 and 2 of Cu2+,ami a diamagnetic form present only in dinucleate complexes of Cu2+ AII can be distin-guished by their spectroscopic properties, especially by electron paramagnetic resonance(EPH) [28,29] .

.5. PREVtoUs EXPEllIMENTAL WOIlK

\Ve have developed a model for studying Cu behavior in biological systems by usingthe copper complex with the synthetic 6-18 peptide segment of the 1'21 Ki-ras oncogenicprotf'in ("6-18 Ki-ras-ru"). This C'omplex <;hows the thr('(' diffprent pa.ram:-..gnetic statesof Cu2+, but when the (,olllplex in solution was in biological conditions (pI! 7-8 and roomtemperature) the paramagnetic behavior of the Cn2+ changes to diamagnctic as can be,ecn in Fig. 1. [30].

156 A"TONIO QUIROZ-GUTIÉRREZ ET AL.

3 ca K

I.-:-----/";'/

I I

FIGURE 1. EPR speetra of the "6-18 Ki-ras-Cu" eomplex in solution. ",here il is possil>le to seethe chango in the magnetic palameters by a ehange in lhe pll.

KI- ra~- Cu proleln somple

)..L=5145 A • T=300K

>-f-¡¡;ZWf-Z

Z<l:;;<la:-,,/\~

-~--------500

STOKES SHIFT ( cm' )

FIGURE 2. Haman vibratioll,,1 spectra oC the "6-18 l\:i-ras.Cu" biorrystal. Th(' :-.trong peal- at700 cm-o) is similar to the as~igned in hemocianin, pl~-u;tociallin él..i'.lIrin and ('stt.lél('janil~ for the O-Obond. sirnililf ¡waks ha.e;,been a.....signed lo a transfcr ehargt' 02 .• eu2+. Tlw ~igllal at 10GO cm-1

fOHnl] on li 111IWlTlo('ianl n ha\'(' h('('n a.•.•s.ignp.; to 2Cu (T 1) 1 h TOIJ¡¿;h ,Ul O hond ¡:\ 1j.

Raman rcsonanre studies uf tht~ sarne complex once crystallizC'd. haw' ShOWIl a clo~esimilarity of lhe "0-18 Ki-ras-Cu" biocrystal witb bltlc cOI'I)('r oxida.se, alld Icl ti, ideIl-tify the Cu-Cu and Cu-O honds, sirnilar bonds have l){,(,1l d('scriL)('d in t he. ppro\'skites.Fig_ 2 la 1).

ELECTHOCHEMICAL STUIlIES ON SOME Cu-Mo INTERACTIONS. J... 157

The Fluoresccnce studies in the same "6-18 Ki-ras-Cu" biocrystal, demonstrate thepresence of Cul+ and at least three different crystalline fields around the coper ion. TheEPH studies of the. same crystal showed the presence of Cu2+ in an orthorombic field [32].AII these findings shows that copper has a behavior in biological compounds as complex

as in superconductors, including a change from paramagnetic to diamagnetic in the copperatoms, the Cu2+ _Cuz t and O-Cu2+ bonding, the geometric arrangements in the crystallinefield, present in this complex, both in solution and in the biocrystaJ.

6. MOLYBDENUM COMPOUNDS IN HUMAN ANO VETERINARY MEDICINE

The interest in studying molybdenum compounds for cancer treatment is not new, Car-ruther and Rcguclson were stlldying molybdates as inhibitors of transplanted tumors inthe sixties. And Mukherjee in India reported evidence of tumor regression in advancedcancer patients following the use of parenteral complexes of phosphotungstic and phospho-molyLdic acids [5,33]. Tetrathiomolibdate has been utilized in veterinary medicine for thetreatment of copper poisoning in sheeps. In which the Cu-Mo complexes are eliminatedby Liie and urine [9].The inhiLitory elfect of Mo on the mammary carcinogenesis in Sprague-Dawley rats,

and the antagonism with the carcinogenic promoting activity of tungsten, has Leen pub-lished [34]. The administration of 10 ppm of Mo in the diet decreased in 50% the carcino-genic elfect of N-nitroso-N-methylurea in the same strain of rats [34). The aflinity of Mocompounds for the estrogen and progesterone receptors (which are specially importantin the development and clinical evolution of experimental animals and human breastcancer), ha"e been used for increasing its concentration in clinico pathological studies [35]and Mo ha"e even Leen used for classification of the estrogen receptors in human breastcancer [36,37].Hecently the inlerest of the National Cancer Institutes in the U.S.A. and sorne Japanese

researches has increased in the produclion of oncostatic drugs containing polymolybdatesin which potenl antitumor activity has been found basically in relation with chemicalmodification of dilferent kind of receptors [34,38]. Phosphomolybdic tungstic acid used in"eterinary medicine ha"e been able to improve dogs with spontaneous advanced malignantlumors [39].i\lore recenlly trtrathiomolybdale has been used for the trealment of Wilson disease in

humans resistant to peuicillamine [40,41 J, aud in patienls with acute neurological symp-loms, lhat often become clinlcally worse when initially lreatrd with penicillamine. Whilelhis paper was under review the group from Ann Arbor Uni"ersity reported lhe results ofsix cases of Wilson's disease treated with tetrathiomolybdate as initial therapy withoutneuroiogical symptoms worsened [.121.

7. CONCLUSIONS

The developiug of molybdenum compollnds for sludying Cu-Mo interactions is a multi-di,riplinary field specially important for the solid state physicist, organic and inorganic

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158 ANTONIO QUIROZ-GUTIÉRREZ ET AL.

chemist, electrochemist and bio-inorganic biochemist. We present in this colloquium ourfirst report on a modified -stabilized Mo(VI)- tetrathiomolybdate synthetized in Mex-ico, for studying sorne Cu-Mo electro chemical interactions, as will be showed in parts lI,m, and IV, (tbis volume).

Since the different Cu-Mo oxidation states interplay depends strongly of the proteinsto which both transitions metals are tightly bound, we designed our experiments using adipeptide with special affinity and well determined chemical bonding with Cu, llsing theHistidil.histidine dipeptide which we found is electrochemically inactive and with specialcapacity to accumulate Cu on the electrode. This can be doubly useful: first it can be usedfor quantitative Cu estimations, even in different Cu oxidation states and second, it canimprove our understanding of Cu-Hist complex in biology, as will be showed in part lI.

Since Cu-Mo interplay is present in many physiological and pathological processesin biology, and oxidation states of both transition metals can be involved in multipleinteractions, we were especially interested in obtaining situations in which one specifiedoxidation state of Mo, will interact with a particular oxidation state of the Cu ions. Gnepossible interactions MoVI-Cul+ using histidine will be showed in part lII. Since bothCu and Mo are electroactives, and histidine is not, and has specific adsorption to bothglassy carbon, and hanging mercury drop electrodes, we study a direct electrochemicalresponse TTM Cu-I1is with the possible formation of one intermediate complex in whichCu(I) may be involve, as will be see in part IV.

The development of Mo compounds is especially important in human and veterinarypharmacology, and it is a priority when this kind of chemicals can possibly be used forcopper poisoning in animals, for Wilson disease, AIDS and cancer treatment in humans.Molybdenum is important for us since Mexico is the third Mo producer in the world [431.

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