Mammalian dihydroorotate dehydrogenase: Physical and catalytic properties of the primary enzyme

ARCHIVES

OF BIOCHEMISTRY

Vol. 191, No. 1, November,

Mammalian

AND

BIOPHYSICS

pp. 23-31, 1978

Dihydroorotate Properties HENRY

Veterans Administration Medicine, University

JAY

Dehydrogenase: Physical of the Primary Enzyme’

FORMAN’

AND

JAMES

and Catalytic

KENNEDY

Hospital, Kansas City, Missouri 64128, and the Departments of Biochemistry and of Kansas, College of Health Sciences and Hospital, Kansas City, Kansas 68103 Received March 8, 1978; revised May 8, 1978

A number of physical and catalytic properties of purified dihydroorotate dehydrogenase from rat liver mitochondria are described. The only potentiahy reducible cofactor detected was iron. The enzyme was also found to contain zinc. The primary enzyme does not contain FAD, FMN, covalently bound flavin, ubiquinone, or labile sulfide. Certain metal chelators were shown to behave as noncompetitive inhibitors of dihydroorotate oxidation and as competitive inhibitors of the reduction of phenaxine methosulfate. The purified preparation can use oxygen as sole electron acceptor, although the reaction rate is relatively slow. The activity of the purified enzyme differs from that of the membrane bound form in a number of ways: the pH maximum is apparently shifted, the effect of thenoyltrifluoroacetone and its K, are markedly changed and the mode of electron transfer to dichlorophenolindophenol is altered. Therefore, only tentative extrapolations to the membrane system regarding activities such as superoxide production can be made.

Mammalian dihydroorotate dehydrogenase is a mitochondrial enzyme which catalyzes the fourth step in de novo pyrimidine biosynthesis. In previous investigations (l-3), we proposed pathways for the transfer of electrons from dihydroorotate to various acceptors based on studies with inhibitors and difference spectroscopy using a particulate preparation. The present report focuses on the nature and catalytic properties of a purified preparation which we refer to as the primary dihydroorotate dehydrogenase, inasmuch as it is homogeneous in subunit composition (4) and is able to oxidize dihydroorotate to orotate with concomitant transfer of electrons to various acceptors. In isolating the primary dehydrogenase, we hoped to be able to extrapolate our results with that preparation back to the membrane system. The results herein give valuable information about which cofactors, notably iron, are actually present in the primary enzyme and which

may be responsible for some of the catalytic functions of the membrane system; however, these studies also indicate that the isolated enzyme shows greater alterations in catalytic properties from those of the membrane preparation than was originally suggested by the prior study of the effects of solubilization by detergents and/or chaotropic salts (5). MATERIALS

AND

METHODS

Soluble dihydroorotate dehydrogenase was purified as described elsewhere (4). Thenoyltrifluoroacetone was obtained from Aldrich Chemical Co. Cytochrome c, DCIP,s l,lO-phenanthroline, and Triton X-100 were obtained from Sigma Chemical Co. Chelex 100 resin was obtained from Bio-Rad Laboratories. Assay conditions are indicated in the figure legends. A modified Zeiss PMQ II or Cary 14 spectrophotometer was used for kinetics studies. Cofactor analyses. For metal analyses, enzyme solutions and reference buffer solutions were passed through chelex 100 resin. Lyophilized samples of purified enzyme were wet-ashed on a hot plate in the presence of 10 ~1 concentrated HSO, with H20:, added

’ Supported by the Medical Research Service of the Veterans Administration. ’ Present address: Department of Physiology, University of Pennsylvania School of Medicine, Philadelphia, Pa. 19174.

’ Abbreviations used: DCIP, dichlorophenolindophenol; thenoyltrifluoroacetone, 4,4,4-tritluoro-I-(2thienyll-1,3-butanedione; PMS, phenaxine methosulfate. 23 0003-9861/78/1911-0023$02.00/O Copyright 0 1978by Academic Press, Inc

24

FORMAN

AND

periodically until ashing was complete. Dried samples were resuspended in 1 N HCl and appropriate dilutions were made for iron and zinc determinations with a Perkin Elmer model 303 atomic absorption spectrometer. Metal standards were obtained from Banco, Inc. through Sargent Welch, Inc. The presence of acidextractable and acid nonextractable flavin was sought using a fluorometric assay (6, 7) with an AmincoBowman spectrophotofluorometer. Samples were assayed for the presence of labile sulfide by the method of Fogo and Popowsky (8) as modiied by King and Morris (9). Samples were assayed for the presence of ubiquinone by the spectrophotometric method of Redfearn (10). Amino acid analyses. The amino acid analyses were performed by the method of Spa&man et al. (11) in a Spinco model 121 analyzer. Samples were hydrolyzed in 5.7 N HCl for 24 h at 105°C in evacuated tubes. After hydrolysis, each sample was dried under reduced pressure in a desiccator. Cysteine was determined as cysteic acid following performic acid oxidation according to Him (12). For tryptophan analysis, samples were hydrolyzed in p-toluene sulfonic acid according to Liu and Chang (13). RESULTS

Characterization

of the Purified Enzyme

In early studies, partially purified preparations of mammalian mitochondrial dihydroorotate dehydrogenases were found to contain ubiquinone, flavin and non-heme iron (14). In membrane preparations, the enzyme has been shown to interact with ubiquinone (14, 15), cytochrome b, and oxygen (l-3). Upon addition of Triton X-100 to the membrane particles, the interaction with cytochrome b is eliminated (3). During purification, enzymatic activity was followed by measuring dihydroorotate-dependent reduction of DCIP and the inhibition of that reduction by superoxide dismutase. Therefore, the various components of the membrane system suspected of being involved with that particular activity were sought in the purified preparation. Table I shows the results of the analyses based on a subunit molecular weight of 61,000 (4). The primary dihydroorotate dehydrogenase appears to be an iron- and zinc-containing protein. FAD and FMN were not found in significant amounts in the purified enzyme. Using the method of Wilson and King (7), fluorometric analysis at pH 3.1 and 7.1 revealed 1.75 x 10e2mol flavin/mol subunit in the acid extractable fraction and no ad-

KENNEDY

ditional flavin upon treatment with excess amounts of proteolytic enzymes. Spectrophotometric analysis, which is not as sensitive but does not require removal of covalently bound flavin, also indicated the absence of flavin in the purified enzyme. In the latter analysis, 0 + 0.1 mol flavin/mol subunit was detected. The last significant amount of flavin is removed during preparative gel electrophoresis, the final puriflcation step (4). Dihydroorotate dehydrogenase in membrane preparations reduces ubiquinone and oxygen directly so that both reduced ubiquinone and superoxide account for distinct portions of the observed DCIP reduction (2, 3). Although kinetic studies with membrane preparations indicated that ubiquinone was probably not an integral part of the primary dehydrogenase (2, 3), the purified preparation was examined for the presence of ubiquinone. The amount of cofactor detected was nil. In studies of NADH-ubiquinone reductase and ubiquinol-cytochrome c reductase, it has been demonstrated that auto-oxidation of ubiquinone and ubiquinol are responsible for most of the superoxide production in mitochondria (16, 17). However, if DCIP is present, it will be reduced by ubiquinone directly as the reaction of DCIP with reduced ubiquinone is faster than the autooxidation of ubiquinone (2, 3). Labile sulfide, which is involved with the active site of most non-heme iron proteins, was not detected. In addition, p-hydroxymercuribenzoate had no effect on enzymic activity. The iron component could

TABLE

I

COFACTOR ANALYSES Cofactor

test

Flavin Labile sulfide zinc Iron Ubiquinone Copper

Mol/mol

subunit


a Limit of method and/or available material. b Multiple analyses were performed on two separately purified enzyme samples. ‘SD.

MAMMALIAN

DIHYDROOROTATE

be bound to a variety of other available including tyrosines, cysteines ligands, and/or histidine moieties. There are in fact substantial amounts of these residues present in the enzyme (Table II). The absorption spectrum of the enzyme (Fig. 1) indicates a broad absorption from the near ultraviolet into the visible range with no obvious peaks. Such a spectrum resembles other non-heme irons proteins, lipoxygenase ( 18)) iron-superoxide dismutase ( 19)) and aconitase (20) where labile sulfide is apparently not present. The iron and zinc are tightly bound as indicated by their ability to remain bound after incubation with and passage through Chelex 100 resin. Preliminary electron paramagnetic resonance experiments indicate resonances around g=2.4 and g=2.0 and none at g=4.3 at liquid nitrogen temperature. Addition of dihydroorotate produced a constantly increasing peak at 280 nm, presumably due to formation of orotate. No other spectral change was noted. The spectrum was not altered by addition of NaBH4. When Triton X-100 was removed by lyophI.00 -

a

25

DEHYDROGENASE

ilization and extraction with petroleum ether and the enzyme redissolved in 0.1% sodium dodecyl sulfate, there was far less absorbance at 275 nm (where Triton X-100 TABLE

II

AMINO ACID COMPOSITION OF DIHYDROOROTATE DEHYDROGENASE Ammo acid

Mol/mol

subunit 40 11 28 48 28 43 50 23 47

LYS His

Arg Asp Thr Ser Glu Pro GUY Ala Val Met Ile Leu

53 42

10 27

TF

61 16

Phe CYS

27 5

Try

1

1

0.75--

oso-cm

.-

0.25..

WAVELENGTH

(nm)

,:LL 330 350 WAVE LENGTH (nm)

1

FIG. 1. (a) Spectra of dihydroorotate dehydrogenase: 0.15 mg/ml soluble dihydroorrotate dehydrogenase in 0.05 M sodium phosphate buffer, pH 7.5,0.05% Triton X-100 (----); 0.15 mg/ml soluble dihydroorotate dehydrogenase in 0.1% sodium dodecyl sulfate (--); 0.02% Triton X-100 (-. -). Spectra were measured using quartz cuvettes, l-cm light path, in a Cary 14 spectrophotometer. (b) Same data plotted on a log-log scale. Lowest line is parallel to base-line for scatter so that deviations from this slope indicate real absorption.

26

FORMAN

absorbs maximally) but a similar spectrum in the near ultraviolet.

AND

broad

Effects of Inhibitors Various metal chelators were tested as inhibitors of the purified dihydroorotate dehydrogenase. Fig. 2 shows the effects of l,lO-phenanthroline and thenoyltrifluoroacetone on the activity at different concentrations of dihydroorotate. These effects were seen regardless of the order of addition. Particulate dihydroorotate dehydrogenase is only partially inhibited by 1 x 10m3M thenoyltrifluoroacetone (2), whereas the soluble enzyme is completely inhibited by thenoyltrifluoroacetone with ZG of 2 x lo-5

M.

Phenazine methosulfate can replace oxygen as an intermediary in electron transport to either DCIP or cytochrome c (3). However, the rate of reduction with cytochrome c is very slow in the absence of PMS. Therefore, in studying the effects of inhibitors as a function of the electron acceptor, the PMS-cytochrome c system was most easily used. Figure 3 shows the effect of l,lO-phenanthroline on dihydroorotate dehydrogenase activity as a function of PMS concentration. The chelator, which is

KENNEDY

noncompetitive with respect to the substrate, dihydroorotate, is competitive with the electron acceptor PMS. The results shown in Fig. 3 are not changed by alterations in the order of addition. Attempts to quantitate the iron content of the enzyme by adding NaBH4 and l,lO-phenanthroline to the purified enzyme produced a change at 510 nm equivalent to only 1 mol Fe per mol subunit in comparison to a standard prepared from FeCb. Addition of larger amounts (in excess of five times the K,) of l,lO-phenanthroline gave no further increase in absorbance at 510 nm. Even if the enzyme is denatured by boiling, the maximum absorbancy corresponds to only about 3 mol Fe/mol subunit. Apparently, the iron is bound very tightly. Charged chelators such as bathophenanthroline sulfonate, ethylenediaminetetraacetate, cyanide, and azide do not inhibit the enzyme. Alterations

in Activity

Membrane bound dihydroorotate dehydrogenase apparently cannot directly reduce DCIP; the magnitude of inhibition of DCIP reduction by superoxide dismutase is

l/V

I

0 FIG. 2. The effect of metal chelators on dihydroorotate dehydrogenase activity. The assay system contained 0.1 M potassium phosphate buffer, pH 7.1, 4.3 x 10e5 M DCIP, 0.05 M KClOd, the indicated concentrations of sodium dihydroorotate, 0.02% Triton X100, 30 pg enzyme and either 5 X 10e4 M l,lO-phenanthroline (0) or 1 X 10d5 M thenoyltrifluoroacetone (0) or no inhibitor (A) in 3 ml at 23% In other experiments where the inhibitor concentrations were varied, 100% inhibition could be obtained. Reduction of DCIP was followed at 600 mn. The rate (v) is in arbitrary units.

I

5

IO 15 lo-'/[PMS]

20

25

FIG. 3. The effect of l,lO-phenanthroline on dihy droorrotate dehydrogenase activity. The assay system contained 0.1 M potassium phosphate buffer, pH 7.1. 2 X 10m5 M cytochrome c, Type VI, 1.67 X 10e4 M sodium ,dihydroorotate, 0.02% Triton X-100, 17 pg enzyme, indicated concentrations of PMS, 0.05 M KC104 and either 5 x lo-’ M l,lO-phenanthroline (A) or no inhibitor (0) in 3 ml at 23%. Reduction of cytochrome c was followed at 550 nm. The rate (v) is in arbitrary units. The Ki for l,lO-phenanthrohne is 4.4 X lo-4 M.

MAMMALIAN

DIHYDROOROTATE

equal to the decrease seen under anaerobic conditions. The remaining activity is likely due to ubiquinone-mediated electron transfer, (3). Superoxide dismutase can completely inhibit DCIP reduction by the soluble purified dihydroorotate dehydrogenase under aerobic conditions (Table III). However, under anaerobic conditions, DCIP reduction by the purified enzyme is equal to that under aerobic conditions and superoxide dismutase does not inhibit until oxygen is allowed to re-enter the cuvette. Oxygen itself, in the absence of DCIP, will serve as a direct electron acceptor from the purified enzyme (Table III). Superoxide dismutase, in the absence of DCIP increased the rate of orotate production of the membrane system (1). Superoxide dismutase, however, did not further increase the rate of orotate production with the purified enzyme, but did still completely inhibit DCIP reduction when DCIP was added. It is therefore surprising that orotate production in the presence of DCIP was TABLE III EFFECTS OF SUPEROXIDE DISMUTASE AND ANAEROBIOSIS ON PURIFIED DIHYDROOROTATE DEHYDROGENASE ACTIVITY’ Sample

DCIP

Air Suu;~-

DCIP reduced

Orotate produced

nmol/min

nmol/min

DEHYDROGENASE

27

not decreased by the addition of concentrations of superoxide dismutase which completely stopped DCIP reduction. This is not a result of allowing or increasing the rate of auto-oxidation of DCIP because the effect of addition of superoxide dismutase at the beginning of the reaction or after much reduced DCIP has accumulated, is the same. Seemingly, the purified enzyme, unlike the membrane-bound form, can transfer electrons directly to DCIP. A further demonstration of the direct interaction of DCIP with the soluble dihydroorotate dehyrogenase is that the K, for DCIP (1.7 x lop5 M at pH 7.1 and 5.8 X 10m6M at pH 7.8) is independent of enzyme concentration. The influence of pH on the activity of purified dihydroorotate dehydrogenase is shown in Fig. 4. The pH optimum of 7.1 differs somewhat from the value of 7.8 for the crude membrane preparation (14). Perchlorate ion, which at low concentrations activates the soluble enzyme, does not alter the pH optimum. The activating effects of chaotropic ions such as perchlorate are apI

dismutase

1

+

+

-

2 3 4 5 6 7

+ + + -+-++ ---

+ -

+ +

2.00 0.05 1.78 1.746

1.89 1.93 1.99 2.096 0.18 0.16 0.00

“Assays for DCIP reduction (600 nm) or orotate production (280 run) at 23” C contained 0.1 M potassium phosphate buffer, pH 7.1, 0.05 M KCIOI, 6.7 x 10m4 M sodium dihydroorotate, 0.02% Triton X-100, and, when present, 8.6 x 10m5M DCIP and/or 3.3 x IO-’ M superoxide dismutase (SOD) in 3 ml. The cuvette used was a slightly modified version of that designed by Hodgson et aZ. (21). Samples were flushed with highly purified Nz (research grade, Matheson) for 20-30 min. ‘When air was allowed to re-enter number 4, the rate slowed gradually (to 0.25 after 20 min) for DCIP reduction but continued at the same rate for orotate production.

?k-+-kPH FIG. 4. The pH maximum for purified dihydroorotate dehydrogenase. The assays contained 0.1 M potassium phosphate buffer at the indicated pH, 4.3 x 10e5 M DCIP, enzyme, 0.17% Triton X-100, 6.7 x 10e4 M sodium dihydroorotate and, either 0.05 M KCIOl (0) or no KClO, (0) in 3 ml at 23°C.

28

FORMAN AND KENNEDY

parently on V,, rather than the K,,, for dihydroorotate. For example, at pH 7.8, perchlorate caused a doubling of I’,,,,, and a 3-fold increase in K, (Fig. 5). Similar results were obtained using the PMS-cytochrome c system with PMS as the variable or using DCIP as the variable in the usual assay system. Lastly, many of the experiments in the earlier literature with whole mitochondria or particles were performed at 37°C. Fig. 6 illustrates the effect of temperature on the rate of dihydroorotate de-

hydrogenase. The AH for activation is 14.9 Kcal. At 37’C, the K,,, for dihydroorotate at pH 7.1 in the presence of 50 m KClO( was 7.7 X lo-6 M. DISCUSSION

Mammalian mitochondrial dihydroorotate dehydrogenase is one of several types of dihydoorotate dehydrogenase which have been described in the literature. These various enzymes differ in both their cofactors and reaction mechanisms. The enzyme responsible for the biosynthesis of erotic acid is mistakenly identified in most textbooks as the inducible NAD-dependent enzyme from Zymobacterium oroticum (22). This soluble enzyme which contains FAD, FMN, and an iron-sulfur center (23) is found only in organisms which grow on pyrimidines. Soluble enzymes which do participate in pyrimidine production are found in Lactobacilus bulgaricus (24) and Crithidia fasciculata (25). Escherichia coli and pseu0 IQ’ domonads contain biosynthetic enzymes [DIH~ATE] which are particulate and which transfer electrons to menaquinone and/or ubiquinone (26-29). It appears that some of these procaryotic types may contain flavin. The C. fasciculata enzyme uses a reduced pteridine as a cofactor. Eucaryote dihydroorotate oxidizing enzymes are located in the mitochondria. The Dxidation of the substrate is linked to the electron transport chain in both Neurospora crassa and mammalian systems (2, 3, 14, 15, 30, 31). A highly purified but nonhomogeneous enzyme from N. crassa In k appears to contain FMN and iron (31). Although this enzyme differs in several other respects from the rat liver mitochondrial enzyme, it was found to be lacking in labile sulfide. Partially purified beef liver dihydroorotate dehydrogenase also contains iron and flavin (14) but this enzyme has 3.20 3.25 never been purified to homogeneity. 3.30 3.30 Whereas the present study has not ruled FIG. 6. Effect of temperA?g<6n purified dihydroorotate dehydrogenaae. Assays contained 0.1 M potaa- out an interaction with a flavin or flavoprosium phosphate, pH 7.1, 4.3 x lo-’ M DCIP, 0.167% tein in the membrane, it appears that the primary dehydrogenase from rat liver miTriton X-100, 3.3 X low4 M sodium dihydroorotate, tochondria contains only iron and zinc as 0.05 M KC104, and 15 wg enzyme in 3 ml at the cofactors (Table I). The spectrum of the indicated temperature. The rate (k) ia in arbitrary units. enzyme does not indicate the presence of

b

MAMMALIAN

DIHYDROOROTATE

either flavin or iron-sulfur prosthetic groups. Fig. 1 does show a broad absorption from the base of the aromatic residue absorbance into the visible range. This is reminiscent of the spectra of several nonheme and nonlabile sulfide containing iron-proteins (18-20). The purified enzyme maintains a rate of superoxide dismutase-inhibitable DCIP reduction which is very close to that of the membrane bound form (4). It appears that the interactions of the enzyme with electron acceptors such as DCIP and oxygen have been altered during the process of purification. Further indications of an alteration of the enzyme site responsible for the release of electrons were also indicated by the changes in the effects of thenoyltrifluoroacetone (Fig. 3). On the other hand, there was no apparent change in the overall rate of nonubiquinone-dependent activity between the membrane bound and purified enzymes. Presumably, the rate-limiting step remains unchanged during purification. Interestingly, changes in the catalytic activity of the active site accompany the solubilization of the enzyme by the nonionic detergent, Triton X-100. Various ions and charged chelators such as azide, cyanide, bathophenanthroline sulfonate, and ethylenediaminetetraacetate are unable to cause inhibition of the enzyme. It may be that the active site, which appears to contain iron, is in a hydrophobic area of the protein. Indeed, much of the soluble enzyme’s surface is apparently hydrophobic as the enzyme requires Triton X-100 for solubility (5). Triton X-100 may make the active site more accessible to compounds such as DCIP and thenoyltrifluoroacetone which are themselves more soluble in nonaqueous solvents than in water. The effects of anions on the activity of soluble dihydroorotate dehydrogenase have been shown to be activation at low concentration (60 mM) and denaturation at higher concentrations with both effects following the well documented series of chaotropic ions (5). Although chaotropic ions usually exert their influence on protein conformation (32), these same ions are potential ligands for iron. The possibility exists

29

DEHYDROGENASE

that the activating effects of low concentrations of these ions on the activity may be due to binding to iron in such a way as to influence the reactivity without altering the binding of the substrates. In solution, the reduction of oxygen by iron can be altered in both rate and mechanism by the presence of different anions (33). Slykhouse and Fee (34) have shown that binding of ligands to some of the coordination sites of iron superoxide dismutase will still allow simultaneous interaction with superoxide. With dihydroorotate dehydrogenase, the rate of the reaction is increased by these ions, whereas the Km’s for dihydroorotate (Fig. 2) and PMS were only slightly altered. The interaction of DCIP and oxygen with the purified enzyme appears to be more than a simple competition for the active site. Although the transfer of electrons through oxygen to DCIP may be faster than the direct reduction of DCIP and would explain superoxide dismutase inhibition in air (Table III), the fact that superoxide dismutase alone cannot increase the rate of orotate production to that seen in the presence of DCIP suggests that a more complicated mechanism is involved. A reaction between a DCIP radical and oxygen and/or superoxide may be involved (Fig. 7), inasmuch as auto-oxidation of fully reduced DCIP has been eliminated as a possibility. Because a one-electron acceptor, such as PMS, can be used by the enzyme, it may be that DCIP accepts one electron, producing an easily reoxidized DCIP radical and simultaneously promoting the reaction of oxygen with the partially reduced enzyme EH2 + DCIPBEH' EH'+02-E+O'+H

+ DCIPH' -

+

2

DCIP + 02' + H+F

DCIPH' + O2

DCIPH' + 02' + H+-

DCIPH2 + O2

SOD '+2H+ 2 O2 + H2°2 + O2 FIG. 7. Proposed mechanism for the co-reduction of DCIP and oxygen by purified dihydroorotate dehydrogenase. EHP, reduced enzyme; E, oxidized enzyme; SOD, superoxide dismutase.

30

FORMAN

AND

to produce superoxide. The superoxide radical then could either react with another molecule of DCIP to produce DCIP radical and oxygen or with a DCIP radical to produce fully reduced DCIP and oxygen. The addition of superoxide dismutase would pull these reactions in such a way as to eliminate the accumulation of fully reduced DCIP without affecting the rate of transfer of electrons from the enzyme itself. This complicated mechanism would account for the differences in the rates of reduction of various one-electron acceptors with purified dihydroorotate dehydrogenase. Both DCIP and 02 react directly with the enzyme but at different rates. Together DCIP and 02 interact in a more complicated way than simple competition so that both direct 0, production is enhanced and DCIP free radical can be reoxidized by O2 with superoxide dismutase then dismuting the resultant 0, . PMS appears to interact at the catalytic site more rapidly than does 02. Reduced PMS then reacts faster with cytochrome c than it does with O2 so that superoxide dismutase does not inhibit PMS-dependent cytochrome c reduction. On the other hand, cytochrome c cannot react directly with the enzyme and its reduction therefore depends upon the production. When DCIP slow rate of 0, or PMS is present, the maximal rate of the formation of orotate is the same. This would best be explained by a mechanism in which the binding of dihyroorotate to the enzyme or reduction of the enzyme by dihydroorotate is the rate-limiting step when the electron acceptor site is saturated. The absorbance change which appeared upon the addition of borohydride and l,lOphenanthroline to active enzyme corresponds to 1 mol of iron per mol of subunit rather than the higher amounts found by atomic absorption. This is probably due to a combination of factors; some of the iron may be inaccessible (an increased absorbance corresponding to 3 mol Fe/mol subunit is seen after boiling the enzyme) and the iron which does react with l,lO-phemmthroline may do so incompletely. The nonenzyme iron complex which was used as a standard is an iron-(1igand)s complex (35). If binding of less than three molecules of

KENNEDY

l,lO-phenanthroline is sufficient to completely inhibit, the spectral change would not, of course, correspond to the nonenzyme-bound complex. It is well known that l,lO-phenanthroline will also bind to zinc. Therefore, we cannot be certain that iron is the site of action of l,lO-phenanthroline; however, iron is the only potentially reducible cofactor which has been identified. A more complete assessment of a functional role for the iron will be carried out using electron paramagnetic resonance. ACKNOWLEDGMENTS The authors thank Dr. James W. Hamilton for amino acid analysis and Dr. Frank 0. Brady for atomic absorption analysis. REFERENCES 1. FORMAN, H. J., AND KENNEDY, J. (1974) Biochem. Biophys. Res. Commun. 60, 1044-1050. 2. FORMAN, H. J., AND KENNEDY, J. (1975) J. Biol.

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MAMMALIAN

DIHYDROOROTATE

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DEHYDROGENASE

31

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146,256-270. 31. MILLER, R. W. (1975) Can. J. Biochem. 53, 1288-1300. 32. HATEFI, Y., AND HANSTEIN, W. G. (1969) Proc. Nat. Acad. Sci. USA 62, 1129-1136. 33. TAUBE, H. (1965) J. Gen. Physiol. 49, (Part 2) 29-50. 34. SLYKHOUSE, T. O., AND FEE, J. A. (1976) J. Biol. Chem. 251:5472-5477. 35. COTTON, F. A., AND WILKINSON, G. (1972) in Advanced Inorganic Chemistry, p. 861, John Wiley & Sons, New York.