T h e w o r k of Steinberg ( 2 5 1 , 252) demonstrated t h a t Aspergillus niger required small quantities of m o l y b d e n u m w h e n g r o w n on a n i t r a t e m e d i u m , w h e r e a s the response to the m e t a l w i t h a m m o n i u m nitrogen w a s considerably less. T h e s e findings provided evidence t h a t m o l y b d e n u m was needed for growth a n d other processes a p a r t from nitrogen fixation. T h e first clear-cut proof of t h e essentiality of traces of m o l y b d e n u m for higher plants w a s demonstrated b y A r n o n a n d Stout (13) i n w a t e r culture experiments w i t h tomato plants. T h e deficiency w a s characterized b y mottling of leaves a n d involution of the l a m i n a e w h i c h w a s prevented specifically b y m o l y b d e n u m , the 19 other elements tested, including v a n a d i u m , h a v i n g n o effect. T h e results w e r e confirmed b y n u m e r o u s workers for other h i g h e r plants including oats (Avena sativa), lettuce (Lactuca sativa), w h i t e m u s t a r d (Brassica hirta [Sinapis alba]), a n d p l u m (Prunus sp.) seedlings, thus demonstrating t h e essentiality of m o l y b d e n u m for p l a n t g r o w t h in general [see reviews b y N a s o n (175) a n d E v a n s ( 6 7 ) ] .
T h e w o r k of M u l d e r (168) confirmed a n d demonstrated a n u m b e r of f u n d a m e n t a l relationships between m o l y b d e n u m a n d nitrogen m e -tabolism i n various organisms. I t showed t h a t Azotobacter a n d Asper-gillus respond m o r e to m o l y b d e n u m w h e n provided w i t h n i t r a t e nitrogen t h a n w i t h a m m o n i u m nitrogen. M u l d e r used the growth-rate curve a n d t h e increasing sporulation of Aspergillus niger w i t h increas-ing a m o u n t s of m o l y b d e n u m to estimate m i n u t e quantities of molyb-d e n u m i n various materials. H e also showemolyb-d t h a t molybmolyb-denum-molyb-deficient tomato plants contained a h i g h concentration of n i t r a t e a n d t h a t denitrifying bacteria g r o w n o n a molybdenum-deficient m e d i u m failed to reduce nitrate.
M o l y b d e n u m has been shown m o r e recently to be essential for the growth a n d cell division of the green alga Scenedesmus obliquus, the molybdenum-deficient cells failing to assimilate n i t r a t e nitrogen. T h e accumulation of n i t r a t e i n plants is n o t specific for a m o l y b d e n u m deficiency since plants deficient i n m a n g a n e s e or sulfur also give rise to t h e same effect [see review b y N a s o n ( 175) ] .
3. Role of Molybdenum as a Component of Nitrate Reductase T h e first step i n n i t r a t e assimilation i n h i g h e r plants a n d in certain microorganisms is catalyzed b y n i t r a t e reductase. Definite proof of t h e
specific involvement of m o l y b d e n u m i n n i t r a t e assimilation b y fungi, higher plants, a n d certain bacteria w a s obtained as a result of t h e isolation a n d characterization of n i t r a t e reductase. T h e e n z y m e w a s characterized from Neurospora crassa a n d soybean (Glycine max) leaves b y N a s o n a n d E v a n s ( 1 7 7 ) a n d E v a n s a n d N a s o n ( 6 9 ) as a sulfhydryl metallo-FAD-protein w h i c h catalyzes t h e transfer of elec-trons from reduced p y r i d i n e nucleotide ( T P N H for t h e Neurospora e n z y m e , T P N H or D P N H for t h e soybean e n z y m e ) to n i t r a t e to form nitrite:
T P N H (or DPNH) + H+ + N 03" -> TPN+ (or DPN+) + N 02~ + H20 I n t h e e a r l y stages of these studies t h e significant sensitivity of the e n z y m e to a n u m b e r of metal-binding agents such as cyanide, azide (but n o t carbon m o n o x i d e ) , potassium ethyl x a n t h a t e , o-phenanthroline a n d 8 h y d r o x y q u i n o l i n e indicated a m e t a l component. T h a t m o l y b -d e n u m is t h e m e t a l component of t h e Neurospora a n -d soybean e n z y m e s was established b y t h e following findings ( 1 8 3 , 1 8 4 , 1 8 6 , 1 8 7 ) : (a) o n l y a m o l y b d e n u m deficiency resulted i n a significant decrease of n i t r a t e reductase in cell-free extracts of Neurospora (Table I ) . E n z y m e activity of molybdenum-deficient Neurospora w a s restored to n o r m a l w i t h i n 1 2 h o u r s after t h e addition of m o l y b d e n u m ; (b) a n increased specific activity of n i t r a t e reductase i n various e n z y m e fractions is ac-companied b y a proportional increase i n m o l y b d e n u m concentration
(Fig. 8 ) ; (c) d u r i n g dialysis of the e n z y m e against a buffered cyanide solution there is a decrease in m o l y b d e n u m content (to one-thirtieth of t h e control) concomitant w i t h a decrease i n e n z y m e activity. Sub-sequent redialysis against molybdenum-free phosphate a n d glutathione to r e m o v e the cyanide from t h e e n z y m e gave no restoration of n i t r a t e reductase n o r a n y increase in t h e negligible m o l y b d e n u m content of t h e e n z y m e ; a n d (d) t h e r e is a specific reactivation of t h e cyanide-dialyzed e n z y m e b y m o l y b d e n u m . T h e addition of m o l y b d e n u m trioxide or sodium m o l y b d a t e reactivated the e n z y m e of 8 5 % of t h e original value. Preincubation w i t h other metals including iron, zinc, m a n g a n e s e , nickel, cobalt, m e r c u r y , tungsten, u r a n i u m , v a n a d i u m , or boron w e r e ineffective in place of m o l y b d e n u m .
I t is of interest t h a t t h e removal of m o l y b d e n u m from the n u t r i e n t m e d i u m results i n a decrease i n n i t r a t e reductase w h i c h is quite dif-ferent from t h e loss of activity brought about b y removal of molyb-d e n u m from t h e purifiemolyb-d e n z y m e , for example, b y cyanimolyb-de molyb-dialysis.
I n t h e latter case t h e inactivated e n z y m e can be restored almost com-pletely b y adding t h e m e t a l back to t h e protein w h e r e a s , i n the case of m o l y b d e n u m deficiency t h e addition of t h e m e t a l to t h e cell-free
T A B L E I
EFFECT OF N U T R I E N T DEFICIENCIES O N NITRATE REDUCTASE I N
CELL-FREE EXTRACTS OF Neurospora crassa* (187)
Treatment + C a - C a + N - Nb + M g - M gb + F e - F e
Per cent growth 100 16 100 8 100 20 100 0.7
Nitrate reductase 26 38 43 5 49 41 29 55
+ C u - C u + Z n - Z n + Mn - M n + Mo - M o + B i o t i n — Biotin
Per cent growth 100 40 100 28 100 30 100 29 100 15
Nitrate reductase 27 79 25 39 30 34 53 10 27 21
β W i l d type 146. Values are units of enzyme activity per milligram protein.
6 Ν and M g were supplied at one-thirtieth and one-hundredth, respectively, of the level in the controls.
488
extract is ineffective. M o l y b d e n u m , therefore, also appears to be neces-s a r y for the adaptive formation of n i t r a t e reductaneces-se in the preneces-sence of nitrate or nitrite b y Neurospora d u r i n g growth, p r e s u m a b l y for t h e synthesis of the protein moiety of the e n z y m e .
It has also been demonstrated t h a t flavin a n d m o l y b d e n u m function as electron carriers in Neurospora n i t r a t e reductase in the following sequence:
T P N H -» FAD (or F M N ) -> Mo -> N O r
T h a t flavin precedes m o l y b d e n u m in the above sequence was indicated b y t h e observation t h a t molybdenum-free e n z y m e cannot catalyze the
200 180 160 o, 140 Ε >»
ö » 120
»•-ο
; | 100 ο σ
£ 80 ο
Ο-œ 60 40
20
5 10 15 μ.§ χ ΙΟ"4 Mo per mg protein
FIG. 8. Relation between molybdenum contents and specific activity of nitrate reductase of various protein fractions from Neurospora (183).
reduction of n i t r a t e to nitrite b y T P N H or reduced flavin. T h e metal-free e n z y m e , however, will catalyze the reduction of F A D or F M N b y T P N H . Addition of m o l y b d e n u m specifically restored t h e ability of t h e e n z y m e to catalyze t h e formation of nitrite from n i t r a t e b y reduced flavin or T P N H or T P N H plus flavin. T h e e n z y m a t i c oxidation of F M N H2 b y m o l y b d a t e u n d e r anaerobic conditions in t h e absence of n i t r a t e suggested t h a t t h e m e t a l was acting as a n electron carrier
[Nicholas a n d N a s o n ( 1 8 4 ) ] . Direct evidence for the role of
molyb-490 A . N A S O N A N D W . D . M C E L R O Y
d e n u m as a n electron carrier was demonstrated b y e x p e r i m e n t in w h i c h reduced m o l y b d a t e p r e p a r e d w i t h N a2S204, e n z y m a t i c a l l y reduces n i t r a t e to nitrite i n t h e absence of added F A D . T h e m o l y b d e n u m appears to be undergoing a n oxidation-reduction reaction from a n oxidation state + 6 to a m o r e reduced level, probably + 5 [Nicholas a n d Stevens ( 1 8 9 ) ] . M o l y b d e n u m forms compounds corresponding to oxida-tion states of + 2 , + 3 , + 4 , + 5 , a n d + 6 .
Similar studies w h i c h identify m o l y b d e n u m as the m e t a l com-ponent, as well as demonstrating t h e sequence a n d m e c h a n i s m of electron transport b y m o l y b d e n u m to be the same as for t h e Neuro-spora e n z y m e , h a v e also been m a d e w i t h n i t r a t e reductase from soy-bean leaves [Nicholas a n d N a s o n ( 1 8 6 ) ] . T h a t m o l y b d e n u m is a com-p o n e n t of soybean leaf nitrate reductase h a s also been indicated b y E v a n s a n d H a l l ( 6 8 ) . T h e properties of a somewhat similar p y r i d i n e nucleotide-nitrate reductase in Escherichia coli a n d its identification as a metalloflavoprotein w i t h m o l y b d e n u m as t h e probable m e t a l component, h a v e also recently been s h o w n ( 1 8 5 ) . Recent evidence b y Kinsky a n d M c E l r o y (125) as well as b y Nicholas a n d Sea w i n
(188) indicates t h a t phosphate is r e q u i r e d for t h e function of the m o l y b d e n u m containing n i t r a t e reductase. These authors suggest t h a t a p h o s p h o m o l y b d e n u m complex is t h e probable electron acceptor from reduced flavin.
H i g h l y purified n i t r a t e reductase from Neurospora is also capable of catalyzing t h e reduction of cytochrome c b y T P N H . I n a n effort to determine w h e t h e r this cytochrome c reductase was the same as n i t r a t e reductase, K i n s k y a n d M c E l r o y (125) studied these two e n z y m a t i c activities w i t h v a r y i n g a m o u n t s of nitrate in the growth m e d i u m . Both activities w e r e induced b y t h e n i t r a t e , a result suggesting t h a t the e n z y m a t i c activities w e r e associated w i t h t h e same protein or t h a t there w a s a d u a l induction b y a single inducer (see Fig. 9 ) . T h e i r results point to two kinds of T P N cytochrome c reductases: ( a ) a constitutive e n z y m e w i t h n o associated n i t r a t e reductase as indicated b y t h e adaptive experiments w i t h a m m o n i a - g r o w n m y c e l i a ; a n d (b) a n e n z y m e associated w i t h n i t r a t e reductase activity. T h e n u m b e r of e n z y m e s concerned i n n i t r a t e reductase a n d cytochrome c reductase activities i n Neurospora a n d h i g h e r plants h a s not been answered con-clusively.
T h e results b y E g a m i , Sato, T a n i g u c h i , a n d associates (see 261) as well as Sadana a n d M c E l r o y (220) indicate t h a t t h e r e a r e different p a t h w a y s for electron transfer for n i t r a t e reduction. T h e s e essentially fall into two general classes called (a) nitrate assimilation, w h i c h represents t h e biological conversion of n i t r a t e to a m m o n i a or to t h e
a m i n o acid or a m i d e level for t h e u l t i m a t e synthesis of nitrogen-con-taining cell constituents such as proteins a n d (b) nitrate respiration in w h i c h n i t r a t e is used b y several microorganisms (for example, Escherichia coli) u n d e r anaerobic or partially anaerobic conditions as a t e r m i n a l electron acceptor in place of oxygen. T h e first step i n n i t r a t e assimilation h a s been characteristically associated w i t h t h e p y r i d i n e nucleotide-molybdoflavoprotein, n i t r a t e reductase, w h i l e the correspond-ing step in n i t r a t e respiration has been indicated to be i n t i m a t e l y in-volved w i t h a cytochrome system possessing n o n - h e m e iron [see review
)J 1 1 l 1 1 ί
-0 4 8 12 16 2-0 24 NO* cone, in medium ( Μ χ ΙΟ3 )
FIG. 9. Effect of nitrate concentration on the adaptive formation of nitrate and cytochrome c reductase activity (125).
b y N a s o n a n d T a k a h a s h i ( 1 8 1 ) ] . T h e respiratory t y p e of n i t r a t e reductase from Escherichia coli, however, h a s v e r y recently been re-ported to contain one a t o m of m o l y b d e n u m a n d forty atoms of non-h e m e iron p e r molecule of e n z y m e ( 1 1 0 , 2 6 0 ) . W non-h i l e tnon-here is n o evidence as to t h e role of m o l y b d e n u m (or iron) in this system, the finding of m o l y b d e n u m in n i t r a t e respiratory e n z y m e lends further support to the general p a t t e r n t h a t h a s emerged, n a m e l y t h a t molyb-d e n u m m a y be a necessary component of all e n z y m e s capable of catalyzing t h e reduction of n i t r a t e to nitrite (for example, the n i t r a t e reductase of t h e assimilatory type, t h a t of the respiratory type, a n d x a n t h i n e oxidase a n d a l d e h y d e oxidase, as indicated in t h e section below on m o l y b d e n u m in a n i m a l s ) .
Evidence t h a t a specific cytochrome as well as iron a r e essential
492 A . N A S O N A N D W . D . M C E L R O Y
components of t h e nitrate-reducing system in Photobacterium fischeri (Achromobacter fischeri) h a s come from nutritional, inhibitory, a n d e n z y m e purification studies. I n these studies it w a s possible to separate nitrate reductase activity from the T P N H ( D P N H ) - f l a v i n reductase ac-tivity. Reduced benzylviologen w a s used as t h e electron donor for n i t r a t e reduction. T h e purified n i t r a t e reductase, however contains a n iron p o r p h y r i n w h i c h is capable of transferring electrons to nitrate.
T h e reduction of this cytochrome component b y T P N H requires the addition of a second protein fraction as well as flavin a n d inorganic iron. F r o m these a n d other studies it is suggested t h a t t h e inorganic iron a n d m o l y b d e n u m determine t h e direction of electron flow as far as n i t r a t e reduction is concerned. It is likely t h a t the iron-containing system is i m p o r t a n t for t h e " n i t r a t e respiration" system of micro-organisms. I t is also possible t h a t future studies will show t h a t Neuro-spora n i t r a t e reductase activity is associated w i t h two proteins, one catalyzing t h e reduction of flavin ( a n d subsequently cytochrome c) a n d t h e second a phosphomolybdoprotein w h i c h is capable of reducing n i t r a t e b y accepting electrons from reduced flavin. T h e a c c o m p a n y i n g scheme (Eq. sequence 4) indicates t h e possible relationships:
T P N H -> flavin -> F e+ ++ -> cytochrome -> 02 (4) nitrate reductase nitrate reductase
(Mo-protein) (Mo-protein) N 03- N 03