I n addition to t h e a p p a r e n t relationship between zinc a n d auxin content i n plants as previously discussed, other biological p h e n o m e n a h a v e been associated w i t h the metal. T h e above-described a u x i n effect possibly accounts for i m p a i r e d flower setting a n d seed production, w h i c h is a conspicuous s y m p t o m of zinc deficiency in high plants. T h e r e a r e reports t h a t in fungi zinc increases the u p t a k e of calcium, phosphorus, a n d m a g n e s i u m as well as t h e efficiency of sugar utilization. Some workers h a v e indicated zinc to be unfavorable for citrate production in fungi, b u t others h a v e reported t h e opposite effect [see Chesters a n d Rolinson ( 4 2 ) ] .
B . C O P P E R
S o m m e r ( 2 4 4 ) is credited w i t h the first demonstration t h a t copper is a n essential element for higher plants. T h e m e t a l has been established as a component of a n u m b e r of different p l a n t enzymes—polyphenol oxidase, monophenol oxidase, laccase, a n d ascorbic acid oxidase.
Enzymatic Role
O n e of t h e general properties ascribed to t h e copper e n z y m e s is the catalysis of the direct oxidation of their substrates b y atmospheric oxygen according t h e equation
Copper enzyme
B H2 + y202 > Β + H20
a n d their failure to function anaerobically [see review b y D a w s o n a n d T a r p l e y ( 5 0 ) ] . T h e i n h e r e n t p r o p e r t y of inorganic copper salts in catalyzing t h e oxidation of various organic substrates b y molecular oxygen w a s discussed previously. Polyphenol oxidase, also called catecholase or tyrosinase, catalyzes t h e oxidation of o-diphenols b y molecular oxygen to form the corresponding quinones a n d w a t e r (Fig.
7 ) , as shown b y t h e early work of Raper. m- or p-Diphenols are n o t
472 A . N A S O N A N D W . D. M C E L R O Y
acted u p o n b y this e n z y m e . Monophenolase, as exemplified b y the e n z y m e from m u s h r o o m , catalyzes t h e conversion of a monophenol to t h e o-diphenol b y molecular oxygen. T h i s e n z y m e has also been called cresolase or tyrosinase; it is always accompanied b y polyphenolase activity, although polyphenolase does not a l w a y s h a v e monophenolase activity. Laccase, w h i c h is obtained from t h e latex of certain species of lacquer trees can oxidize p-diphenols a n d o-diphenols to the correspond-ing quinones a n d water. Ascorbic acid oxidase catalyzes t h e oxidation of ascorbic acid to form dehydroascorbic acid a n d w a t e r (see Fig. 7 ) .
FIG. 7. Over-all reactions of tryosinase and ascorbic acid oxidase.
T h e activities of these oxidases are dependent on t h e copper content, w h i c h i n purified preparations ranges between 0.1 a n d 0 . 3 5 % . T h e copper is tightly b o u n d a n d cannot be removed b y dialysis against water. However, t r e a t m e n t w i t h acids or cyanide a n d subsequent dialysis result in t h e removal of copper from t h e protein. Reconstitution of these e n z y m e s has been accomplished b y adding back copper. T h e copper oxidases a r e inhibited b y a n u m b e r of m e t a l binding agents, carbon monoxide inhibiting the phenol oxidases of potato a n d m u s h -room b u t not laccase. Inhibition b y carbon monoxide is not reversed by light i n a g r e e m e n t w i t h t h e properties of copper-carbon monoxide models.
T h e best evidence thus far for t h e m e c h a n i s m of action of cop-per w a s presented b y Kubowitz (132, 133). Using potato polyphenol
oxidase, h e obtained data indicating t h a t copper is concerned in electron transport a p p a r e n t l y b y undergoing cyclic oxidation-reduction between C u ^ a n d C u+ d u r i n g t h e e n z y m a t i c transfer of electrons from sub-strate to oxygen. H e showed t h a t 1 mole of o-diphenol reduced t h e C u+ + e n z y m e to t h e cuprous form, yielding 1 mole of o-quinone. I n the re-action of a molecule of o-diphenol, 2 cupric atoms a r e reduced to t h e cuprous ( C u+) form as the carbon monoxide complex, the u p t a k e of car-bon monoxide being determined manometrically. I n the presence of one molecule of o-diphenol, 2 cupric atoms are reduced to the cuprous form, since one molecule of carbon monoxide is b o u n d to two atoms of phenolase copper. T h i s constitutes the best evidence t h u s far for a n oxidation-reduction role of copper at t h e e n z y m a t i c level. T h e copper of laccase, b y analogy to polyphenolase, p r e s u m a b l y acts in the same m a n n e r .
T h e monophenolase activity w h i c h often accompanies polyphenolase activity has been a controversial a n d much-discussed subject. T h e facts t h a t t h e ratio of t h e two activities is easily altered a n d t h a t added copper readily exchanges w i t h the enzyme's copper d u r i n g poly-phenolase action, a n d to a lesser extent d u r i n g monopoly-phenolase action, suggest t h a t t h e two activities a r e i n d e p e n d e n t of one another. T h i s does not exclude t h e possibility t h a t these two activities a r e due to two dif-ferent sites on t h e same protein molecule a n d t h a t each m a y r e q u i r e
copper as a prosthetic group. I n support of this idea a r e t h e observations t h a t t h e two activities a r e associated w i t h t h e same electrophoretic a n d ultracentrifugal component a n d t h a t t h e y are similarly inhibited b y t h e same metal-binding reagents a n d competitive substrates [see re-views b y D a w s o n a n d T a r p l e y (50) a n d M a s o n ( 1 5 6 ) ] .
T h e recent experiments of Dressier a n d D a w s o n (57, 58) h a v e shed further light on this question. B y studying the exchange between radioactive C u6 4 cupric ions a n d resting (nonfunctioning) tyrosinase (purified from t h e m u s h r o o m Psalliota campestris) as well as the catalytically functioning e n z y m e t h e y obtained data w h i c h support t h e hypothesis of two distinct activity centers, i.e., monophenolase (cresolase) a n d polyphenolase (catecholase) sites. T h e i r results sug-gested t h a t copper is firmly b o u n d to t h e tyrosinase protein a n d t h a t t h e resting enzyme essentially does not undergo exchange w i t h radio-active cupric ions except w h e n the e n z y m e contains copper bonded at inactive sites, i.e., o n l y w h e n the e n z y m e is i m p u r e or partially in-activated. However, w h e n the e n z y m e w a s actively catalyzing the oxidation of polyphenols a n exchange between t h e copper of the e n z y m e w i t h ionic C u6 4 occurred depending on the a m o u n t of substrate employed (on t h e n u m b e r of o-dihydric phenol molecules oxidized)
474 A . N A S O N A N D W . D . M C E L R O Y
a n d on t h e t y p e of tyrosinase preparation used. Of the two general types of tryosinase preparations, the one h a v i n g a low molecular weight a n d a catecholase to cresolase activity ratio of about t w e n t y or higher
(called " h i g h catecholase" e n z y m e ) incorporated m u c h m o r e radio-active copper d u r i n g catalysis of t h e oxidation of o-dihydric phenols t h a n t h e " h i g h cresolase" e n z y m e t y p e (high molecular weight a n d a catecholase to cresolase activity ratio of about ten or l o w e r ) . Dressier a n d D a w s o n (58) h a v e concluded, on t h e basis of the above experi-m e n t s , t h a t t h e copper at t h e functioning catecholase activity sites is exchangeable whereas t h a t at t h e cresolase sites is nonexchangeable.
T h e y feel t h a t t h e catecholase sites a r e little, or n o t at all, involved i n t h e oxidation of monophenols a n d t h a t t h e oxidation of a monophenol b y tyrosinase m a y not proceed via a n o-dihydric phenol.
A n opposing viewpoint has attributed monophenolase activity to a n o n e n z y m a t i c , copper-catalyzed reaction. Kertesz ( 1 2 3 ) , studying the m e c h a n i s m of phenolase oxidate of m o n o h y d r i c phenols w i t h a purified potato e n z y m e , reported t h a t additions of C u+ + determined further in-creases i n monophenolase activity; a n d t h a t cobalt, v a n a d i u m , a n d nickel could replace copper, though less effectively. H e interpreted these data to m e a n t h a t t h e monophenolase activity of tyrosinase w a s d u e to free metallic ions w h i c h accelerated t h e n o n e n z y m a t i c reaction between o-quinones a n d m o n o h y d r i c phenols as follows:
o-dihydric phenol (polyphenolase) o-quinone (1) free
o-quinone + monohydric phenol -f H2O * 2o-dihydric phenol (2) metal
T h u s h e contended polyphenolase or tyrosinase to be a complex system composed of a n o-dihydric phenol (or o-quinone), a n e n z y m e specific for o-dihydric phenols, a n d free metallic ions, t h e latter catalyzing the spontaneous reaction between o-quinone a n d monophenols (e.g., tyro-sine). As shown b y Eqs. 1 a n d 2, the second phenolic group of dihydric phenol arising from the monophenol should come from water. T h i s viewpoint, however, has been v e r y effectively refuted b y t h e recent experiments of Mason, Fowlks, a n d Peterson ( 1 5 7 ) . T h e y used 02 1 8
a n d H201 8 in separate experiments a n d found t h a t all the oxygen introduced as t h e h y d r o x y l group in t h e b e n z e n e r i n g of monophenol derivatives b y t h e action of phenolase (tyrosinase) c a m e from molecular oxygen. I n other words, monophenolase is a n e n z y m e belonging to the broad group of oxygen transferases [see review b y M a s o n ( 1 5 6 ) ] .
T h e question concerning t h e existence of a t r u e ascorbic acid oxidase e n z y m e has been raised from t i m e to time. Although it h a s been suggested t h a t t h e catalytic activity of ascorbic acid oxidase m a y
be attributed to traces of ionic copper resulting from a n ionization of copper b o u n d to nonspecific protein m a t t e r or other colloidal material, t h e present evidence is heavily against this view (49, 5 0 ) . T h e w o r k of D a w s o n a n d his colleagues (49, 50) w i t h ascorbic acid oxidase also suggests a m e c h a n i s m of a C u+ +- C u+ cycle for this e n z y m e . Using C u6 4, t h e y w e r e able to show a n exchange between the copper of ascorbic acid oxidase w i t h ionic copper o n l y w h e n t h e e n z y m e w a s actively catalyzing t h e oxidation of ascorbic acid. N o exchange took place i n the resting e n z y m e , i n the e n z y m e inactivated d u r i n g the course of the reaction, or in t h e absence of oxygen even w i t h substrate present. T h e copper of ascorbic acid oxidase, w h i c h is initially i n t h e divalent state, p r e s u m a b l y shuttles reversibly between t h e divalent a n d monovalent forms d u r i n g e n z y m e catalysis. T h e fact t h a t both ascorbic acid a n d oxy-gen a r e necessary for t h e C u+ +- C u+ reaction of t h e e n z y m e , suggests the formation of a n i n t e r m e d i a r y t e r n a r y complex of oxygen, ascorbic acid, a n d e n z y m e . I n model systems t h e u s u a l t y p e of covalent complex
formed b y Cu** is one w i t h a coordination n u m b e r of 4 h a v i n g a square coplanar configuration for t h e directed valences of t h e copper atom ( 2 8 9 ) . C u+ w i t h a coordination n u m b e r of 4 exists in a tetrahedral con-figuration. Since square coplanar bonds a r e considered to be m u c h stronger t h a n t e t r a h e d r a l bonds ( 1 9 9 ) , copper exchange would be m o r e likely to occur w h e n t h e copper bond is i n t h e tetrahedral configura-tion. T h e report (112) t h a t covalent nickel compounds of the square p l a n a r t y p e do not exchange w i t h radioactive nickelous ions i n solution, whereas t h e t e t r a h e d r a l nickel complexes w h i c h a r e of a weaker bond strength do, tends to support t h e above hypothesis. T h e copper e n z y m e s m a y h a v e a significant role i n t e r m i n a l respiration of some plants although additional evidence is necessary in support of this function.
B e n h a m o w et al. (26) observed t h a t t h e reaction inactivation of functioning ascorbic acid oxidase w a s greatly increased i n t h e presence of free cupric ions w h e r e a s t h e nonfunctioning e n z y m e w a s unaffected.
T h i s h a s led t h e m to suggest t h a t C u+ + inactivation involves chemical groupings (possibly — S H ) w h i c h a r e present (or n o t exposed) in the resting or nonfunctioning e n z y m e a n d w h i c h become exposed w h e n t h e e n z y m e functions.
T h e light blue color of purified ascorbic acid oxidase is probably d u e to its copper content. T h i s is v e r y likely also t h e case for hemo-cuprein, a copper-bearing protein insolated from the r e d blood cells of m a m m a l s . O n t h e other h a n d t h e phenolase studied b y Kubowitz (132, 133) h a d a faint yellow color a n d showed n o outstanding absorption other t h a n t h a t of t h e characteristic tyrosine-trytophan spectrum in t h e ultraviolet region. H e m o c y a n i n , a copper-protein complex found in
476 A . N A S O N A N D W . D . M C E L R O Y
the blood of certain invertebrates, exists n a t u r a l l y in t h e cuprous condi-tion a n d is blue w h e n oxygenated a n d only faintly colored w h e n de-oxygenated. It w a s thus presumed at one t i m e t h a t the blue color of h i g h l y purified laccase w a s a p r o p e r t y of the copper b y analogy w i t h the blue color of the oxygenated h e m o c y a n i n s a n d hemocuprein. It w a s later shown, however, t h a t t h e blue color w a s d u e to a carbohydrate-containing component free of protein a n d copper. I t can be seen from these examples t h a t t h e relationship between color a n d valence state of t h e copper in t h e various copper proteins is not entirely clear. T h e possible role of these copper-containing oxidases in t h e respiration of plants is discussed in another chapter of this series.
M o r e recently a n e w addition has been proposed to t h e list of copper enzymes. M a h l e r a n d co-workers (152) reported t h a t purified samples of k i d n e y uricase contain 0 . 0 5 % copper. A n earlier report h a d im-plicated uricase as a zinc e n z y m e (221 ) .
T h e original suggestion of Keilin a n d H a r t r e e (117) t h a t cytochrome oxidase of a n i m a l tissues contains significant quantities of copper ( a n d iron) has n o w been confirmed b y n u m e r o u s workers (60, 148, 196).
Moreover, a n u m b e r of investigators h a v e observed t h a t animals sub-jected to a copper-deficient diet showed a conspicuous decrease in cyto-chrome oxidase activity (44, 76, 109, 2 9 8 ) . Purified cytocyto-chrome oxidase preparations contain one a t o m of copper per a t o m of iron per mole of cytochrome a, a proportion w h i c h is m a i n t a i n e d at all stages of puri-fication (90, 224, 2 5 8 ) . However, t h e r e is no c o m m o n a g r e e m e n t a m o n g workers i n this area as to t h e state a n d function of copper in this e n z y m e system. Sands a n d Beinert (223) using p a r a m a g n e t i c resonance spectrometry obtained data w h i c h are consistent w i t h t h e idea that t h e copper of cytochrome oxidase is reduced b y electron-donating sub-strates of t h e purified e n z y m e , a reduction w h i c h is specific a n d possibly connected w i t h t h e function of the system. A similar view is held b y Griffiths a n d W h a r t o n (90) a n d T a k e m o r i ( 2 5 8 ) . T h e latter has indicated t h a t the copper i n cytochrome a exists in cupric form i n contrast to t h e results of V a n d r a a n d W a i n i o ( 2 7 2 ) , w h o reported t h a t t h e copper of cytochrome oxidase is mostly i n the cuprous state a n d firmly b o u n d to the e n z y m e . T h e inhibition of cytochrome oxidase b y alleged copper-chelating agents such as ferrocyanide, salicytal-doxime, e t h y l x a n t h a t e (258) a n d bathocuproine sulfonate sodium salt
(or 2,9-dimethyl-4,7-diphenyl-l,10-phenanthroline sodium disulfonate) (297) has been contradicted b y t h e experiments of Griffiths a n d W h a r t o n ( 9 0 ) , w h o w e r e u n a b l e to observe a n effect b y t h e latter compound. Yonetani (297) has concluded t h a t although t h e copper in oxidized cytochrome oxidase is in the cupric state and is
en-zymatically reducible (corresponding to 6 0 - 7 0 % of t h e total copper), it does n o t participate in t h e electron transfer system of cytochrome oxidase. Griffiths a n d W h a r t o n ( 9 0 ) attribute Yonetani's results to a contamination b y ionic copper a n d contend t h a t copper is involved in cytochrome oxidase activity b y being firmly b o u n d i n a specific configuration so t h a t it is not vulnerable to copper-chelating agents.
Asano ( 1 5 , 1 6 ) has reported the separation of a n e n z y m a t i c nitrite-reducing system from a Micrococcus strain into a soluble component w h i c h is activated b y C u+ a n d C u+ + ions, a n d a particulate component w h i c h is e n h a n c e d b y F e ^ a n d F e+ + +.