L inorg, nucl. Chem. 1975. Vol. 37, pp 1503-1509.

Pergamon Press, Printed in Great Brflain

Bio-inorganic Section

BIOINORGANIC CHEMISTRY OF OXYGEN

EI-ICHIRO OCHIAI
Department of Chemistry, University of British Columbia, Vancouver, B.C., Canada V6T 1W5

(Received 6 August 1974)

Abstract--A wide variety of reactions of oxygen involved in biological systems is discussed, and an attempt is made
to group the reactions into a set of rather simple reaction patterns. Thus, a coherent understanding of the reaction
mechanisms of oxygen may be made to emerge from the discussion.

REACTIVY OF OXYGEN O2-(free) is very reactive for kinetic as well as ther-

(I) Bonding nature in oxygen modynamic reasons.
Figure 1 shows the orbitals involved. The ground state The O-O bond cleavage of H202 could occur in two
of 02 (sYg) has the configuration: KK(o's)2(o'*)e(crp) 2 ways: (6) and (7) (Table 1); the former is a little more
(0r~)2(~rx)20r*)~(0r*)t. The outermost portion of O_,- is favorable thermodynamically than the latter.
(m)~(m)2(p f):(~.,), (m):(Tr*)2 is equivalent to (pz° )2(p/)~
where a and b refer to oxygen atoms a and b; and (3) Reactions of oxygen in biological systems
(o-s):(¢*)2= (s")2(sb) :. Therefore, in O2- the electronic They are summarized in Table 2.
configuration about oxygen atom includes (s)(py)(p~),
which forms s f hybrid orbitals. Two of the sp 2 orbitals TRANSPORTATION AND STORAGE OF OXYGEN
are occupied by lone pairs and the electron in another of
Oxygen molecule binds reversible with transition metal
s f is shared with that on the other oxygen atom. The complexes. Since oxygen is strongly electronegative
bond of 02:- is maintained by (¢rp)2. Another electron (oxidizing), the binding would be accompanied by at least
added to 022- would go into the antibonding orbital o'*,
partial electron transfer to oxygen from the transition
and the molecule would split into O' and 02 .
metal. A thermodynamic argument about the oxygen
binding to transition metal complexes was outlined in a
(2) Thermodynamic and reactivity of oxygen and related previous paper[2]. The two possible modes of oxygen
compounds adduct are:
The relevant data are compiled in Table 1. Table 1
indicates the difficulty in O-O bond cleavage of O2
molecule at ordinary temperature and in the first M + O2,~-~MO2(M+Of) (9)
reduction of 02. The latter is due to the pairing energy in 2M + O:~M:O~ ((M+)2022-). (10)
7r* orbitals and is the main reason for the inertness of 02
molecule, in spite of the fact that 02 in the ground state is The examples are:
a biradical. For example, in the following reaction (8), the MO2: M=Fe(II): hemoglobin, myoglobin, model
energy gain in the formation compounds [3-5]
M = Co(II): Co(salen)B, etc. [2, 6]
•02" + C=C--, .O-C-C-O. (8) M202: M = Fe(II): hemerythrin[7]
M = Cu(I) : hemocyanin[8], tyrosinase[9], etc.
of C-O bond is not enough to compensate the energy M = Co(II): Co(salen)B, etc.[2, 6].
required for the opening of the double bond and for the
pairing energy. This may be described [1] as an example of (1) M02
spin-forbiddenness of reaction; i.e. Offtriplet)+ Recent successes in synthesizing model
C=C(singlet)~ spin-forbidden. However, Offtriplet) can compounds[3-5] for myglobin and X-raying an oxyge-
readily react with a radical (doublet) such as H' or R' nated model compound[10] at last seem to have solved
(alkyl or aryl), because the bond formed would release the problem of oxygen bonding structure, which had long
energy enough to compensate the pairing energy. A been defying an unequivocal determination. The structure
similar situation would be obtained with Co(II) complexes of the oxygenated model compound[10] is sketched in
and Fe(II)(high spin) complexes; in these cases O2--M "t Fig. 2. This is the structure proposed first by Pauling[I 1].
will be obtained as the result of the electron coupling. The last outstanding problem is the mechanism how the

1503
1504 EMcHmo OCHIAI

Table 1. Thermodynamicdata of oxygenreactions field effect. This effect causes a transition of the electron
originally located in the highest d-orbital d~y into d~-r~.
Reaction aH(kcal/mole) AG(kcal/mole) The resultant electronic configuration can be described
essentially by Fem ~-O2-, with a weak ~'-interaction
(1) O2~20 +119 between d~ and ~r* which pairs up the two electrons. The
(2) 1/21-I2+ O5-~I-IO2 +4.9 +9.3
(3) 1/2H2+ HO2~ H202 -37.4 -32.8 situation is essentially the same as that in (1:1)COO2
(4) 1/2H2+ H202~ HO + H20 - 16.1 -23' 1 complex[13, 14], though in the latter case the interaction
(5) 1/2H2+ HO~ H20 -67.1 -62.7 of d~ and ~r* leaves the unpaired electron in its
(6) H202~ H20 + O +34.2 +24.2 antibonding level[13].
(7) H202~2HO +51.0 +39.6 Essentially the same electronic configuration is seen in
the 02 complex of tryptophan dioxygenase[15], that of
cytochrome P-450[16], and that of peroxidase, the
so-called compound III[17].

(2) M202
The electronic and bonding structures of oxyhemeryt-
hrin and oxyhemocyanin are yet to be established, though
many circumstantial evidence suggests that they are
Fig. 1. Orbitalsin O2-moiety. essentially (Fem)20~2- and (CunhO~2-, respectively.
The structure of the model Co-complexes has been well
established [2, 6]; it is essentially LCom~O22- ~ CoH~L
o/° with some minor contribution of ~r-bonding.

/-4:7 I
B
The exact bonding structure of oxyhemerythrin and
oxyhemocyanin are under intensive investigation and
much debate. The two metal ions involved in the
oxyproteins have been revealed by various spectroscopic
Fig. 2. The bonding structure of a model Fe-O2 complex as methods to be located close enough to give rise to a
elucidated by X-ray crystallography(10):<Fe-O-O = 135°, dis- certain degree of antiferromagnetic spin-spin coupling
tance (O-O)= 1.23-2"26A. between themselves. Possible structures are:

/0--6- o ~ °-
M*-. . . . . . --~M* ..0-..
2-0 \O
(A) (B) (c) (13) (E)

diagmagnetic oxyhemoglobin is formed from O2(3Es) and Structures (A-C) have a feature in common that 0, inserts
the high spin Fe(II) complex. This problem was discussed in between two M's whereas in structures (D,E) 02 binds
by us in a previous paper[12]. The process includes an with only one of M's. Proposals (C-E) are based on a
edgewise approach of 02, and the coupling between the strong antiferromagnetic coupling observed in ox-
odd electrons in It* and ir*~of 02 and those in dz2 and dzx yhemerythrin and methemerthrin derivatives; it is attri-
of Fe(II) complex. These interactions (coupling) are buted to M-O-M bond. Proposals (D,E) for ox-
bonding in nature, making the coordination reaction of 02 yhemerythrin came from M6ssbauer data[7] that the two
encounter no major potential barrier. As well established, Fe(III)'s are not in equivalent environment. Some authors
Fe is located a little below the porphyrin ring in the deoxy maintain that structure(C) can also explain the dissimilar-
state and it moves into the center of the plane upon ity. The structure (A) is the one guessed at from Co-model
O2-coordination, thus becoming to feel a greater ligand complexes but seems to have been abandoned for

Table 2. Reactionsof oxygenin biologicalsystems

(1) 02+ M~M02 hemoglobin, myoglobin

02 + M2~--M202 hemerythrin, hemocyanin
(2) 202-~O2 + 022- superoxide dismutase
(3) 02 + SH--~SHO2 dioxygenase:tryptophan dioxygenase
metapyrocatechase,protocatechuate-
3,4-dioxygenase,etc.
(4) O2+ SH + AH2--,SOH mono-oxygenase: cytochrome P-450-
+H20+A dependent mono-oxygenases,
tyrosinase, etc.
(5) SH ~ dehydrogenation--~ oxidases: cytochrome c oxidase, laccase,
O2(~ H202 or -~2H20) ascorbate oxidase, etc.
 Bioinorganicchemistryof oxygen 1505

oxyhemerythrin by most workers, Structure (A) might not also been indicated spectroscopically in the case of
be entirely inconsistent with the data, if the dissymmetry protocatechuate-3,4-dioxygenase[20]. The 7r-bonding in
of hemerythrin molecule is taken account of. O2--Fem seems to be insignificant in oxytryptophan
Tyrosinase has recently been shown[18] to behave very dioxygenase, unlike oxymyglobin. Thus 02-. would show
analogously as hemocyanin; the optical spectrum of a rather prominent radical character, as is inferred from
oxytyrosinase was found to be almost identical with that the effect of superoxide dismutase[21]. This radical O2-'
of oxyhemocyanin. An interesting reaction found[18] for could readily attack a double bond. Co(salen) which is
tyrosinase may be written as follows: known to form Com-O5-' has been shown [22] to catalyze
the tryptophan dioxygenase reaction (12).
+H202
E-(CuU)2 ~ [E-(Cun)2(H205) ~.~ E-(CuII)2022-] The mechanism to be proposed for nonheme Fe(III)
( Tr form ) I T" form) enzymes (intradiol dioxygenase) is shown in Fig. 3. In a
(T' form)
recent paper[20], the oxidation state of Fe in the
(11) enzyme-substrate (ES) complex was taken to be +3,
E-(Cu~h + 05
(T" form) because of its optical spectral similarity to that of Fe"I-E;
the EPR data, however, strongly indicated that Fe(III)
The spectrum of T" form is the same as oxyhemocyanin; was reduced upon addition of the substrate. We propose
this indicates that oxyhemocyanin may have such a that the optical spectrum for the ES complex is due to the
structure as can be derived from (CuU)2 and 025-. No semiquinone coordinated to Fe~I-E as written in Fig. 3. If
indication has been obtained to suggest a M-O-M bonding this hypothesis is correct, the mechanism is consistent
in oxyhecyanin and oxytyrosine. Therefore, the pos- with all the data and observations. For Fe(II) enzymes
sibilities are (A) and (B). Structure (B) was suggested for (extradiol dioxygenase), a reasonable mechanism includ-
oxyhemocyanin by Mason et al.[19]. ing ESO2 has been proposed [23]. The difference between
the two mechanisms is due to the redox potential of Fe in
Dioxygenase (02 activation) the enzymes; in the intradiol dioxygenases Fe(III) is the
Three examples illustrate the reaction pattern[20]: more stable form than Fe(II), whereas in the extradiol

/CH2CH--COOH
tryptophan dioxygenase
-~-CH2~H--COOH (Fe(ll)-heme)
(12)
NH~ v "N
H H~CHO
OH
pyrocittechase

~
(Fe(lll)-nonheme)
OH
+ O~ (13)
OH

O
II OH

~ OH

OH
+ 02
metapyrocatechase
(F©(lI)-rtonhem¢) _C C/==O
t~C--oH
(14)

The oxygen addition occurs across a C=C double bond. 02 dioxygenases the opposite is the case. This difference
addition to C=C double bond is known to occur would also cause a variation in the coordination of the
photochemically and one of the initial products is substrate to Fe-site in the two classes of enzymes.
dioxetane. Dioxetane is usually unstable due to bond
strains and liable to decompose to two carbonyls. This Mono-oxygenase (0-0 bond cleavage, Mode I)
scheme seems to fit nicely to the enzymatic reactions. The A general equation of mono-oxygenation is expressed
only objection [1] comes from an estimated endothermic- by:
ity of the formation of the dioxetanes.
The oxygen molecule must be "activated" by the en- R£-H+O2+AH2~R3C-OH+H20+A (15)
zymes. Tryptophan dioxygenase is a heme enzyme and was
shown [15] to form an oxygenated intermediate in the pres- A variety of enzymes catalyze this type of reaction; they
ence of the substrate. The optical spectrum of the oxy- are: (a) cytochrome dependent mono-oxygenases[16, 24]
enzyme is very similar to that of oxymyglobin, indicating such as steroid l l/3-hydroxylase and alkane mono-
that the bonding structure of oxytryptophan dioxygenase oxygenase;
is essentially the same as that of oxymyoglobin; i.e. (b) Cu-enzymes: tyrosinase [9,18] and dopamine-/3-
O2-Fefn. A ternary complex: enzyme-O2-substrate has hydroxylase [25];
1506 EMCHmOOCHIAI

(c) metal-free flavoproteins such as p-hydroxybenzoate One famous mechanism by Mason[27] assumes a still
hydroxylase and imidazoleacetate mono-oxygenase. The unidentified oxocopper complex (Cut),O. Our proposal
Eqn (15) indicates that O2 is reduced to 022- state (Fig. 4) is based on the same ground discussed above and
whatever the form might be, and O:2- splits into "O" and the oxygenated species mentioned earlier.
O2-(H20). The most famous theory[l] coined the name
"oxenoid" to such a species as represented by "O". The Peroxidase (0-0 bond cleavage, Mode 11)
present author[12] indicated that "O" is of the character The primary intermediate in the reaction of perox-
of O(tD), the first excited state of O-atom. Peroxides such idase-peroxide (ROOH, R = H or alkyl) is represented by:
as M-O--O2-, HO2- and CF3CO--O--O- can react with
nucleophilic reagents; for example, E-Fe IIl+ -OOR -* [E-Fe m ~ O-O-R]. (22)

---CO--O--O- + N ~ , .6,

H O 2 - + I- ) H O - + OI- (17)

That is, the terminal oxygen atom in these compounds As far as the oxidation state of the system is concerned,
tends to react as an electrophile. If the electrophilicity is this is equivalent to E-FemO22- in the cytochrome P-450
strong enough, the oxygen atom may react with a weak system. However, the reaction is dramatically different.
base such as ~r-electrons of a C--C double bond or even The difference is due to the attachment of R ÷ to 022-
the tr-bond electron of C-H; e.g. moiety; this makes such a splitting as to lead to the

J
CF2CO--O---O- + ~..C=C~ ~C \O)C /x.
) CF3COO- + 1- (18)

CF~CO--O--O- + " CFaCOO- + HO--~ (19)

These are exactly the same types of reaction which formation of "O" difficult[12]. Another mode of O-O
cytochrome P-450 dependent mono-oxygenases catalyze bond cleavage now becomes feasible.
and O(~D) performs [26]. Therefore, a likely
mechanism[12] of cytochrome P-450 dependent mono- [Fem ~O-O-R ] ~ [Fe 1" *--O.(FeW-O2)]+ .OR.
oxygenase's reactions is: (23)
+02 +e
E-Fe n >E-FEm-O2- . ) E-FelII-O22- The R O. radical thus formed oxidizes the porphyrin ring
in the cases of horse radish peroxidase [28] and catalase,
:,") C and a protein residue in the case of cytochrome c
+c-. , E-Fe . . . . _--O/+-I peroxidase [29].
H
Reduction of 02 to 2H20(O-O bond cleavage,Mode 111)
E-FeUI-OU-+ / C This reaction is effected by cytochrome c oxidase,
> O\H tyrosinase, laccase, ascorbate oxidase and ceruloplasmin.
(20)
The minimum requirement for this category of enzymes
Thermodynamically, the reaction can be broken down seems to be the presence of a set of two metal ion centers
into: closely located. That one such set is involved has been

FelII_O22- F e U I - - O 2- + O : X

~ H + O ~ O H :AH = -107 kcal (21)

We have no estimate for x, but it would not be very much established for copper enzymes [30]. Such a two-metal
different from +34 kcal (for H 2 0 2 ~ H20 + O). Hence, the center (MonMbm) binds 02, effectively reducing it to 052-:
reaction as a whole should be amply exothermic. The (M,n+tMb=÷l)o22-. If this 052- is released as HOC, the
reaction mechanism, however, is most likelY of SN2type; in system may not be very effective in reducing it further to
other words, O(~D) would not be liberated as such. 2H20. In such an ineffective system catalase would be
The mechanism of tyrosinase is far from understood. required as a cofactor. Examples include FADH2,
 Bioinorganic chemistry of oxygen 1507

H H~ copper ions found in active hemocyanin and tyrosinase.

All relevant data and observations fit to the mechanism to
O--r be proposed (Fig. 5). In Fig. 5, Cu(a)~Cu(b)~Cu2(c)
indicates the sequence of the internal electron transfer,
and the process in [ ] represents the internal electron
Fe~z E 02
transfer. Thermodynamically speaking, Cu2(c) group has
the highest reduction potential; therefore in a reductive

I3:il _ i
"---k
~....{/o~/0
.p.o
"-0
H
\ ~
~ ~,

Fig. 3. A mechanism for the enzymatic activity of Fe(III)-

~Q'O
~.0
U /I

H
"8 ~
Z::~

(ESO2)
titration of Rhus laccase[31], Cuz(c) is the first to be
reduced. However, it is known that the blue Cu(a) is the
site where the electron goes into first. In fact, in another
reductive titration[30] with Polyporus laccase with
Cu(b) blocked, the Cu(a) is the first to be reduced. O~
binds to Cu2~(c) in the fully reduced enzyme, being
nonheme dioxygenases.
reduced to 02>; additional two electrons are then
supplied by Cu~(b) and CuX(a). The most interesting and
complicated system is cytochrome c oxidase. It has now
been well established[32] that the working unit of the
C,J ~Cu Cuz 2H + enzyme contains one cytochrome a, one cytochrome a3
fd~ : '
-,.co~ : dJ and two copper atoms. The two copper atoms have
H
different properties. The EPR spectrum of one of them,
Cu(a) is normal while that of another Cu(fl) is abnormal
0 2HO OH
0 e ON in that the spectrum is very readily saturated and is not
L observable under ordinary conditions. The magnetic
F..,_~_.c,~ ._~ e . \ c ~,,_o 2H+ co~._o\2H2e+
" I II i'a / 2 @ 4""4--- iu I 2@ . • i suceeptibility of the whole enzyme becomes maximum at
' % : , . ~ O + C u ~-0 Cu 4-0 Cu~ 0
H about 50-75 per cent reduction. This magnetic behavior of
the enzyme and the abnormal EPR of Cu(/3) have led
~ - OH some[32] to hypothesize an antiferromagnetic coupling
21..I+ between Cu(/3) and cytochrome a3. A popularly held
CuI~ OH Cu~_O ,,.2 Q sequence[32] of the internal electron transfer is shown
':= ' JZ, ~ o by:

,°0 cyt. a -~Cu(a)~cyt. m

Fig. 4. A mechanism for tyrosinase activity: one (middle portion) ..'¢ !. (26)
is for catecholase activity and another (bottom portion) is for
cresolase activity (mono-oxygenation). These are not entirely satisfactory in view of the data and
the observations remarked above. Our proposal is
pyridoxamine and others as the substrate of the reaction summarized in Fig. 6. The dotted line indicates the
of the type: antiferromagnetic coupling. When two electrons added
(50 per cent reduction), Cu(/3) becomes diamagnetic and
SH2 + 02 ---+S + H202. (24) hence, the antiferromagnetic coupling would be lifted. As
a result, the magnetic moment of cyt. a3 (high spin) would
However, in the metalloenzymes mentioned above, it be fully manifested. The addition of oxygen may occur on
appears that 02 > is kept coordinated to (Mo"+'Mb" <) until (a3)Fe"... CuI(fl) as shown in Fig. 6; this was suggested
two more electrons are added to the system: by Bennett [33].

m a n- 14_..O m n+l

2e, 4H-
+ 2H20. (25) Cu(b) • "~ ~Cu(b) ~ Cu(b),Cu(b)

/~/m--I C@ C ) Cu2( c ) Cu2( c ),~ 2( c )

M j ~~ O b

Tyrosinase (catecholase activity) has already been men-

tioned (Fig. 4). The electrons come from the substrate
catechol. The so-called "blue" oxidases (laccase, ascor- 2H +
bate oxidase and ceruloplasmin) contain [30] three differ- c~(o) 4H* 0 C~Co)4# ZAH. aA% C~(o/
ent types of copper: "blue" Cu(Cu(a)), "non-blue"
Cu(b) : CuZ(bi : Cu(U)
Cu(Cu(b)) and EPR-non-detectable Cu2(c). The last group tu:( ~'z
of copper is such that the coppers involved are situated in c )q;- c~e(c ) L?~c)
proximity and are magnetically coupled with one another; Fig, 5. A mechanism for laccase, ascorbate oxidase and
in other words, this group is very similar to the set of ceruloplasmin.
1508 EI-ICHIROOCHIAI

Table 3. Reaction patterns of oxygen

Fundamental pattern Examples of reaction Examples of enzyme

(i) O~ ~ 02- Fe n + 02 ~ Fe m''''02- hemoglobin, myoglobin

Fe" + 02 ~ Fe m - 02-- O2-activation: dioxygenase
• , 2t
(it) 0 2 ~ 02
2-
(Fell)2 + 02 ~ (Femh022- hemerythrin
(Cu2h + O~ ~ (CunhO~2- hemocyanin, tyrosinase
Fe m - 02- + e ~ Fe TM- O22- cytochrome P-450
(iii) O~2-~ 0 + 02-(H20) Fe m - 022- + SH ~ Fe m - O2- cytochrome P-450-dependent
+ SOH hydroxylases
(O--O cleavage Mode I) (CulI)2022- "4"SH ~ (Cu"hO 2- tyrosinase
+ SOH
(iv) 022- + e ~ .O- + O2-(H20) Fem-OOR ~ [Fe ~v ~ O ~ ] peroxidase
+ .OR
(O--O cleavage, Mode II)
(v) O22- + 2e ~ 202-(2H20) CuIIO22-Cu" + 2e -->Cu~H+ 202- tyrosinase, laccase, etc.
(O-O cleavage, Mode III) Cu"O~2-Fe m + 2e ~ Cu" . . . . Fem cytochrome-c oxidase
+ 202-

9. A recent review, W. H. Vanneste and A. Suberbuhler, In

Molecular Mechanisms of Oxygen Activation, loc. cit., p. 371.
10. J. P. Collman, R. R. Gagne, R. A. Reed, W. T. Robinson and
a~c~(#)'c~(a
%i'= ) ~e Ila~ curB>*
IL i 3 c~(a)Tnternol
&: ' Im ~ C ~ / 038'i m) ~ Cul(a
elec*ron
~rGnsfer G. A. Rodley, Proc. Nat. Acad. Sci. (USA) 71,1326 (1974).
11. L. Pauling, Nature 203, 182 (1964).
12, E. I. Ochiai, J. lnorg. Nucl. Chem. 36, 2129 (1974).
k 2°-'2H°' " ~ 13. E. I. Ochiai, J. Inorg. Nucl. Chem. 35, 1727 (1973).
14. B. M. Hoffman, D. L. Diemente and F. Basolo, J. Am. Chem.
Soc. 92, 61 (1970)•
a-~C~(~')--PCu (~) a-*Cu~-j3~C u (a)
15. O. Hayaishi, Y. Ishimura, T. Nakazawa and M. Nozaki, In
I~ .02 Biochemie des Sauerstoffs (Edited by B. Hess and Hi.
Staudinger), p. 1%. Springer, Berlin (1%8).
16. A recent review, V. Ullrich, Angew. Chem. Internat. Edn. 11,
701 (1972).
a-~,,Cu(~.w,Cu;a) ~, i ~.Cu Cu(e) q
Conform-
a,.~Cu( 4'Cu(e
17. J. B. Wittenberg, R. W. Noble, B. A. Wittenberg, E. Antonini,
M. Brunori and J. Wyman, J. Biol. Chem. 242, 626 (1%7).
18. R. L. Jolley, Jr., L. H. Evans and H. S. Mason, Biochem.
a~ at'ionoI ~ j
3 change Biophys. Res. Commun. 46, 878 (1972).
19. T. H. Moss, D. C. Gould, A. Ehrenberg, J. S. Lier and H. S.
Fig. 6. A mechanism for cytochrome c oxidase. Mason, Biochemistry 12, 2444 (1973).
20. H. Fujisawa, M. Uyeda, Y. Kojima, M. Nozaki and O.
Hayaishi, J. Biol. Chem. 247, 4414 (1972); H. Fujisawa, K.
CONCLUSION Hiromi, M. Uyeda, S. Okano, M. Nozaki and O. Hayaishi,
The reaction patterns discussed above are summarized ibid. 247, 4422 (1972)•
in Table 3. 21. F. Hirata and O. Hayaishi, ibid. 246, 7825 (1971).
22. A. Nishinaga, T. Itahara and T. Matsuura, 28th Nat. Meeting
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