Copper(II) Schiff-base complexes and apoglobin stability

Bioclmmi~ry ELSEVIER

Journal of Inorganic Biochemistry73 (1999) 137-144

Copper(II) Schiff-base complexes and apoglobin stability Joseph J. Stephanos a.,, Leia M. Jackson b, Anthony W. Addison b a

ChemistryDepartment, Facultyof Science, El-Menoufia University, Shebin EI-Kom, Egypt bChemistry Department, Drexel University, Philadelphia, PA 20104, USA

Received 29 May 1998; received in revised form 6 November 1998; accepted 7 January 1999

Abstract

N,N'-Propylene-bis-(N-salicylidene) copper(II) (Cu(Salpm)) explicitly stabilizes apomyoglobin. The optical spectrum of this copper(II) Schiff-base complex of apomyoglobin arises from the electronic excitations of ~r*-O-Salprn --)d~2_y2 and N-Salpm ---)dx2_.~.2.Shifts of these transitions with respect to those of the parent complex may be a consequence of hydrophobic solvatochromism or binding of an additional ligand. ESR parameters imply no change in the identity of the first coordination sphere around the copper, while hydrophobic solvatochromism cannot be excluded. Combination of copper(II) Schiff-base complex with apomyoglobin does not inhibit the ability of apomyoglobin to extract hemin from the main component of Glycera dibranchiata hemoglobin. Hemin replaces the copper complex, and the value of the apparent first-order rate constant varies with time. The mechanism involves dissociative and associative interchange pathways. Values of rate constants for transfer of hemin to copper(II) Schiff-base apomyoglobin complex, as well as the change of concentration with time are evaluated. © 1999 Elsevier Science Inc. All rights reserved. Keywords: Schiff bases; Copper complexes; Apoglobin stability

I. Introduction

Iron protoporphyrin IX, which is designated as heme (Fe 2+ ) or hemin (Fe 3+ ), serves as the prosthetic group for a vital category of proteins and enzymes known collectively as hemoproteins. Although the prosthetic group of hemoproteins is the active site, apoprotein selects, specifies and regulates the reactivity, and also inhibits auto-oxidation [ 1 ]. Dissociation of the prosthetic group of hemoprotein yields unstable apoprotein [2]. To target factors influencing the stability of the embedded prosthetic heme, transfer of hemin from Aplysia californica myoglobin to horse heart apomyoglobin was meticulously investigated [ 3 ]. The kinetics fit a triangular mechanism for a biphasic reaction. A

A, * Corresponding author.

Neither heme nor hemin is released from deoxy, oxy, carbonyl or azide derivatives of the Aplysia californica, or when the distal His E7 of the apohemoprotein is replaced by leucine or valine. This suggests a role for hydrophobicity of the active site and for a trans influence of the axial ligand in determining the thermodynamic stability of the embedded prosthetic group [3]. Furthermore, deletion of the D-helix from 13subunits of human hemoglobin or myoglobin provides flexibility in the region between the C- and E-helices, and gives an explanation for the fragile hemin binding [4] of or-hemoglobin subunits and Glycera dibranchiata hemoglobins [ 5,6]. Finally, the missing salt bridge between the propionate of hemin and the Phe cD3 residue is also an important factor in determining the stability of the prosthetic group [7-9]. In the absence of heme, apoprotein has compact and unique spatial structure with an extended hydrophobic core in solution with a pH close to neutral [ 10, I 1 ]. The conformational structure of apomyoglobin ( A p o M b ) has maximum stability at about 30°C, and breaks down reversibly with high velocity either upon heating or cooling [ 10]. Stability of hemoprotein and apoprotein may be described by [ 10,12-14]

0162-0134/99/$ - see front matter © 1999 Elsevier Science Inc. All rights reserved. PHS01 62-0134(99)00007-0

J.J. Stephanos et al. / Journal of lnorganic Biochemistry 73 (1999) 137-144

138

Native (N) 11°'13) Apoprotein Fe 2+ tl/2 = 30 days 60% helical Fe 3+ t]/2 -- 1-2 days hemin Partial unfolding

Hemoprotein( t 2 ]

- Heme /

Intermediate (I) (]4] Molten globule Unfolded (U) Unfolding of B and E Complete loss of "--Complete disruption the heme binding sites

To understand the role of the prosthetic in the stability of apoglobin, we attempt to investigate apomyoglobin' s recognition of metal complexes and the consequent influence on stability of apomyoglobin and affinity toward hemin.

KPi buffer pH 7, 50 mM KPi buffer pH 7, and 0.1 M KPi buffer pH 7. After dialysis, the sample was centrifuged to remove any precipitate.

2. Materials and methods

Preparations of N,N'-propylene-bis-(N-salicylidene)copper(Ii) (Cu(Salprn)) and other Schiff bases like Cu(Nalprn) [22-26], Cu(SenN-pn) [27,28] and M(Cyclops)ClO4 ( M = C u ( I I ) , Ni(II)) [29] have been previously described.

2.2. Preparation of metal(ll) Schiff-base complexes

Isolation of the main component of Glycera dibranchiata hemoglobin (Hbc) was based on a previously published procedure [ 15,16]. The buffer used throughout was pH 7 potassium phosphate buffer (0.1 M KPi, p, = 0.2). Other reagents used were supplied by Sigma, Aldrich and Fisher. Fractional distillation was used for purification of methyl ethyl ketone. Proteins were assayed as iron(II) carbonyls, using the pyridyl-hemochromogen method [ 17 ]. Electronic spectra and kinetic data were digitized using a Perkin-Elmer Lambda-3B spectrophotometer interfaced with an Apple Macintosh SE computer through an analog connection MC interface (STI Computer Instrumentation and Controls). ESR spectra were obtained at 77 K on a Varian E-12, X-band spectrometer, calibrated at 77 K on diphenylpicrylhydrazyl. ESR spectra were simulated based on SIM 14/14a and adapted to the Macintosh platform [ 18,19 ].

Cu ( Salprn )

Cu ( Nalprn )

2.1. Preparation of apomyoglobin (ApoMbhh) [20,21] Horse heart myoglobin (Mbhh) and 1.4 mole equivalent of potassium ferricyanide were dissolved in a few ml of 5 mM potassium phosphate buffer, KPi pH 7, and stirred for 15 min at 4°C to oxidize Mbhh(II)O2. A Sephadex G-25 column was used to separate Mbhh (III) HzO, and the pH value of Mbhh (III)H20 was lowered to 2.1 using 1 M HC1. Methyl ethyl ketone was used to extract the hemin from the myoglobin (three extractions). The remaining polypeptide was dialyzed for 1 h at 4°C, in each of 0.1 M NaHCO3, H20, 20 mM

Cu (SenN-pn) ÷

M (Cyclops)CI04 M =Cu(tl)sNi(II)

2.3. Preparation of N,N'-propylene-bis-( N-salicylidene )copper(II) apomyoglobin complex (Cu(Salprn)(ApoMbhh)) ApoMbh~ was mixed with ] .4-fold excess of Cu(Salprn), then stirred for 30 min and run through a G-25 Sephadex

Table 1 Electronic excitations of Cn (Salprn) and Cu ( Salprn ) ( ApoMbhh ) in the UV region Complex

(O-Salprn) ~d~2 .... [31 ]

(O-Salprn) ~dx2 ~-~[31]

(N-Salprn) ~d~-, £ [32,33]

Cu (Salprn) '~ Abs. (nm) e,(mM I cm-I)

355 10.5

270 12.5

232

328 6.7

257 16

214 25

Cu (Salprn) (ApoMbhh) h Abs. (nm) e'(mM Icm

1)

J Dissolved in a minimum amount of DMF then diluted with 0.1 M KPi, pH 7 [26]. b In 0.1 M KPi, pH 7.

J.J. Stephanos et aL / Journal of Inorganic Biochemistry 73 (1999) 137-144

column. Binding of Cu(Salpm) to ApoMbhh was confirmed spectroscopically.

A

3. Results

Electronic and X-band ESR spectra of Cu(Salprn) and Cu(Salprn) (ApoMbhD are summarized in Tables 1 and 2. Other attempts to prepare M(L)(ApoMbhD have failed, where M(L)=Cu(SenN-pn)CIO4, Cu(Nalprn) and M(Cyclops) C104 (M = Cu(II), or Ni(II) ). Combination of Cu(Salprn) and ApoMbhh did not inhibit the ability of ApoMbhh to extract hemin from Hbc(III). Fig. 1 pictures time course difference spectra of Cu (Salprn) (ApoMbhh) andHbc(III) versusHbc(III).Themaximamonitor the formation of metMbh,, while the minima detect the collapse of Hbc (II|) due to hemin dissociation. The persistent peak at 355 nm characterizes free Cu(Salprn) (Fig. 1). An initial shift in the isosbestic points at 390 nm indicates the presence of intermediate. The optical absorption spectrum of Cu(Salprn)(ApoMb,h) is given in Fig. 2. When Hbc(III) and excess Cu(Salprn)(ApoMbhO are mixed at 25°C and pH 7, the absorption at 408 nm steadily increases over time (Fig. 3). The initial concentration ratio (1:14 molar ratio) between the two proteins justifies a pseudo first-order treatment. If Mb,h(Ill) H20

&,'p

7.6 I~M {c.(sa~pm}}(ao,,M,,~, tl Ilk5 m M

7.1 tzM yield =

+

0.3

0.2

|:~

o.1

0.0

-0.1

~ v d e a g ~ (urn) Fig. 1. Time course (30 rain intervals) difference spectra of Hbc(III) obtained by adding Cu(Salprn) (ApoMbhh) in the sample cell and a compensated amount of buffer to Hbc(III) in the reference cell in 0.1 M KPi, pH 7.0, 25°C, scale: - 0.3:0.5. The maxima at 408 nm and minima at 390 nm correspond to Sorer bands of Mbh,(II1) H20 and Hbc(III), respectively, and the persistent peak at 355 nm characterizes free Cu (Salpm). !

Hbc (III)

139

Cu ( Salprn )

-i

decomposes with time

,

,

,

!

!

r~

V-T-t-T ..... i - - - , T -

94%

then the apparent 'first-order' rate constant is obtained from experimental data as In( [Absl,-[Abs]~ ~

k

where [Abs],, corresponds to the absorption of the initial concentration of Glycera dibranchiata hemoglobin (7.6 IxM, e4o8 ,m = 92 raM-J cm-~), and [Abs[, is the absorption at time t. The spectrum of the final product characterizes Mbhh(III)H20. [Abs]~ is the final absorption of (Mbhh(III)H20) (eaos,,m = 188 mM i cm-1). The value of k~r,p varies with time (Fig. 4). Similar behavior was noticed in the heroin transfer either from cytochrome-cpe~vxidase

;

10

i

i

: I

-2 . . . . .

Table 2 First-derivative ESR parameters of Cu (Salprn) and Cu ( Salpm ) (ApoMbnh) Complex

g:

g,

g.~

gll

A II

Cu(Salprn) b Cu ( Salprn ) (ApoMbhh) ~

2.418 2.398

2.325 2.346

1.976 1.976

2.137 2.146

247 260

In units of 1 0 - 4 c m - ~ . "Cu(Salprn) dissolved in a minimum amount of DMF then diluted to I mM with 0.1 M KPi, pH 7, at 77 K. 1 mM ofCu(Salprn) (ApoMbh,} in 0.1 M KPi, pH 7, at 77 K.

200

3O0

i

-

400

(nm) Fig. 2. Optical absorption spectrum of Cu(Salprn) (ApoMb,,) at pH 7, 25°C.

[30] orAplysia californica myoglobin [3] to apomyogiobin, suggesting a similar mechanism (Scheme 1). Under pseudo first-order conditions the kinetics follow: This mechanism consists of one reversible and two parallel step reactions. Considering the initial concentrations of

140

J.J. Stephanos et al. / Journal of lnorganic Biochemistry 73 (1999) 137-144

t.,

(a) 42-42~{~--O" ta _ta _.r~ -

i~.Oll)

4~- ~ . - 4 3 - - - [ 3 - - 4 3 - - - L 1

I "

/

L_

m.ldnl~# 0m~tmn~)

~tm~r) . . . . . . . . . . . . . . .

m.tmm~o Scheme 1.

[Hb~(III) ] , + [ H b c ( I I I ) H 2 0 ] . + [Mbh.(III)H20]t nn

. . . . . . . . 0

q-- . . . . . . . . . . . 200

4-

-

-4-

400

. . . .

¢. . . . .

600

800

-I

= [ concentration ] iota,

1000

Time (rain)

ts (b) L4

and

~ n , ~ r i : i m m ~

I

d [ Mbhh (III) H20 ] d[Hb¢(III) ] + d[Hbc(III)H20] + =0 dt dt dt

~

The exact solution of the differential equations:

0

2000

4000

6000

8000

10000

12000

14000

Time (mitt)

Fig. 3. Absorbance-time data sets at 408 nm at 25°C, and pH 7, when 7.6 }xM of Hb~(III) is mixed with 0.106 mM of Cu(Salpm) (ApoMhbh), and the fitting of triangular kinetic scheme for the hemin release from Glycero dibranchiata to Schiff's base copper(II) apomyoglobin: (a) time scale 0:1000 rain, (b) time scale 0:14000 rain.

[Hb~(III) ] = [Hbc(III) 1o [Hbc(III)H20 ] = [Hbc(III)H20]o [ Hbc(III) ]o ~= [Hbc(III)H20]o [ Mbhh (III) H 2 0 ] = 0 then [ Hb~ (III) ] o + [ Hb~ (III) H20 ] o = [ concentration ] tota~ and at any time. t:

J.J. Stephanos et al. / Journal of lnarganic Biochemistry 73 (1999) 137-144

141

ot = 0.5(kj +k_~ + k2 + k3) fl=0.5{ (k, + k _ , +k2 + k 3 ) 2 - 4 ( k ~ k 3 + k _

lk~ +k3k2) }0.5

The optical absorbance at any time t is governed by Eq. ( 4 ) [3]:

[ Abs ], = eMbtmm2o( [ Hbc (III) ] o + [Hb~(III)HzO]o) + {n cosh fit m + ~ sinh fit} exp( - at)

(o) (4)

I

where n = [ Hb~(III) ]o(e~bo~m) - eMb~I.m2o) m = ~°Hbc(lll)[

Hbc(III) ]od + e.t, (m)H2o[Hb~(III) H20] o f

- e~(d[Hb~(III) ]o + [Hb~(III)H20]o)

3400

3000 2600 H(G) Fig. 5. First-derivativeESR spectraof (a) Cu(Salprn) dissolvedin a minimum amountof DMF thendilutedto 1 mM with0. I M KPi,pH 7, at 77 K, (b) 1 mM of Cu(Salprn)(ApoMbh,) in 0.1 M KPi,pH 7, at 77 K.

4. Discussion • CuN202

m

The optical spectrum of Cu(Salpm) is well defined as sharp and broad bands at 355 and 270 nm, respectively, arising from the electronic excitation of the ar*-O-Salprn orbital to dx2_y: of Cu(II) [31]. Another sharp band at 232 nm results from the electronic transition N-Salprn--~d~2_y2 [32,33]. The positions of these ligand-to-metal charge transfer (LMCT) transitions indicate blue shifts in the electronic spectrum of Cu(Salprn) (ApoMbh,) with respect to those of the parent complex, Cu(Salprn) (Table 1). The shifts to higher energy may be a result of introducing a fifth ligand into the coordination sphere [ 34], or a consequent solvatochromism in which the band shifts to the blue with a less polar environment [35,36]. The ESR spectrum of Cu(Salprn) (Fig. 5) indicates that copper ion is ligated to an equivalent pair of nitrogen nuclei and shows a maximum coupling along the perpendicular direction, and a minimum coupling along the parallel direction (Table 2). A similar spectrum is obtained for Cu(Salprn) (ApoMbhh) (Fig. 5). Cu(Salprn) and Cu(Salprn)(ApoMbhh) show a hyperfine splitting of four features. The parallel component (A M) appears well resolved, and relatively higher in Cu(Salpm)(ApoMbh,) than in Cu(Salprn). The tendency for All to increase and g, to decrease with decrease in the degree of tetrahedral distortion has been reported previously [37-40]. As illustrated [41], there is a good linear correlation between A u and gv and the tetrahedral distortion directs that trend. Therefore, All or gll may be used as an index to the electronic properties of Cu(Salprn) and Cu ( Salprn ) (ApoMbhh). Correlation between A ~ and g IIcan be observed (Fig. 6) for Cu(Salpm), Cu(Salprn)(ApoMbhh) and related Schiff-base copper(II) complexes of chromophore CuN202, derived from 13-diketone, diamine and o-hydroxybenzaldhyde [26]. The ESR parameters of Cu (Salprn) (ApoMbhh) show slight deviation. However, the

o Cu(Salprn) • Cu(Salprn)(ApoPIb ..................

) hit

All x I 0 4 c m - i

m~

2.13

;LIE5

2.18

~

2.23

g.

Fig. 6. Relationship betweenA ~and g~ for CuO2N2chromophore.

F test indicates that the F table value exceeds the experimental value, and the deviation is not statistically significant. Consequently, there is no change in the identity of the first coordination sphere, CuN20 2 chromophore, around the copper of Cu(Salprn) (ApoMb,,). The induced tetrahedral distortion of Cu (Salprn) may be a result of the hydrophobic interaction of ApoMbhh, as suggested by the electronic spectrum. The hydrophobic vacancy of the heme pocket, which contacts His FS, is known to accommodate a variety of organic and inorganic compounds such as l-anilino-8-naphthalenesulfonate [42], cyclopropane, pyridine [43,44], N2, Xe, AuI3 and H313- [45-48]. The recognition of ApoMbhh is stereoselective and very sensitive to shape, size and electronic state. Comparing the structure of Cu (Salprn) with reference to that of hemin shows an agreement of the main moiety. Gel filtration chromatography (Sephadex G 25) or dialysis shows that Cu(Salprn) is bound to ApoMbhh strongly enough to survive at least these mild treatments. Consequently, binding within a hydrophobic pocket cannot be excluded. The constant presence of the peak at 355 nm (Fig. 1) indicates that the hemin replaces the copper complex. Unlike Cu(Salprn) (APOMbh,), [Com(acacen) (NH3)2] + irreversibly replaces the hemin in metmyoglobin [49].

J.J. Stephanos et al. / Journal of lnorganic Biochemistry 73 (1999) 137-144

142

Table 3 The k values calculated from IAbs],= ~ub,nl.~o([Hb¢(III) ]o+ [Hbc(III)H:O]o) + {n cosh ~t+ (m/fl) sinh fit} exp( - at), for the absorption-time curve in reaction of Hb~(IIl) and ApoMbhh in presence and absence of Cu (Salpm) at 25°C in comparison to those of Mb~p~(Ill) and ApoMbhn [3] Holodonor

Hb~(Ill)

Hb~(III)

Mb~p~(III) ~

Holoacceptor

ApoMbhh

Cu (Salpm) (ApoMbh.)

ApoMb..

4.88 1.24 2.75 1.43 92 h 190 ~ 188

78.37 39.44 64.36 2.944 81 ~ 120 r 188

103 k I (min -I ) I 03 k_ l (min - J) 104 k2 (min -~ ) 104 k~ (min -~ ) ~A (mM i cm-i) eA' (mM - ~cm - t ) e~,n~l m)n2o (mM- 1cm- ~)

+0.01% ± 0.03% ___0.1% +0.004% -+0.03% -+0.03% + 0.03%

3.23 2.08 8.61 6.35 92 b 77.3 ~ 188

rg

± 0.00l

0.97

0.23

Aplysia californica myoglobin. r, Hb~(Ill). Mb,pt(III ) H20. d Hbc(III) HzO. Cu (Salpm) (Mbhh(IIl) H20). Mbapt(llI). gr=A/(A+A').

Fig. 3 represents the a b s o r p t i o n - t i m e c u r v e o f the h e m i n transfer f r o m H b c ( I I I ) to C u ( S a l p r n ) ( A p o M b n , ) , at 25°C and p H 7. The kinetics fit a triangular m e c h a n i s m for a biphasic reaction (Fig. 3). N o n l i n e a r least-squares fitting to Eq. (4) yields ce = 0.003272 + 6.4 X 1 0 - 5 , / 3 = 0.0031023 + 3.3 × 10 - s , n = - 1 . 4 0 0 0 9 7 _ 3 . 3 × 10 - 3 and m = 0.00299444 _ 4.3 × 10 - 5 ( S S E = 0.00216). Refitting c~,/3, n and m gives the values of k and the extinction coefficients, ( T a b l e 3). Similar to previous findings [30,50,51 ], the lack o f rate d e p e n d e n c e upon the concentration o f A p o M b h , suggests slow release o f h e m i n f r o m Aplysia californica m y o globin, while the absence o f heroin aggregation in the absence or presence o f Cu ( S a l p r n ) indicates the fast reconstitution to freshly prepared ApoMbhh. The formation o f the aquated species [ H b c ( I I I ) H 2 0 ] is m u c h slower in c o m p a r i s o n with a similar process forAplysia californica m y o g l o b i n [ 3 ]. To detect the identity o f the intermediate, A ' , h e m i n transfer f r o m Glycera dibranchiata h e m o g l o b i n to ApoMbnh was f o l l o w e d , under the same conditions. Hb~(llI) penta-coordinate 3.z3×~o2 r a i n ~4o8 ~,~= 92 mM - J cm ~-~ ~ 2.08x10 2rain kz = 8.61 × 10 4 min ~ 97%

I

I

Hbc(llI)H20 hexa-coordinate ~4o8..1 = 77.3 mM - ~cm k3= 6.35 × 10-4 rain3%

A l t h o u g h the majority o f Glycera dibranchiata h e m o g l o bin has penta-coordinate c o n f o r m a t i o n ( 9 7 % ) , which is c o m parable to the p r e v i o u s finding [ 5 2 ] , the extraction rates of h e m i n f r o m hexa- and penta-coordinate forms are not far apart. Therefore, it is likely that the h e m e transfer follows S c h e m e 2. Since the calculated extinction coefficient o f A ' ( 1 9 0 m M - ~ c m - ~ ) is c o m p a r a b l e to that o f M b h h ( I I I ) H 2 0 ( 188

m M - ~c m - ~), and does not match that o f Hbc (III) H 2 0 (77.3 m M -~ c m - ~ ) , it is reasonable to propose C u ( S a l p r n ) ( Mbhh (III) H 2 0 ) as an intermediate. Hbc(lII) 92 mM- i cm- I k2=2.75 × 10- 4 min -I

4.gBX 10-2min

~408 nm =

I

Cu(Salpm)(Mbhh(llI)H20)

-9' 1.24× 10- 2 rain

I

eaog,n, = 190 mM - ~cmk 3 = 1,43 × 10 -4 min-

Fig. 7 represents the time d e p e n d e n c e o f the concentrations of [ H b c ( I I I ) ] , C u ( S a l p m ) ( M b h h ( I I I ) H 2 0 ) and [Mbhh(III)H 2 0 ] . The m e c h a n i s m i n v o l v e s two pathways: firstly, dissociative interchange through the k2 pathway, where h e m i a , enters the h y d r o p h o b i c cavity o f ApoMbhh i m m e d i a t e l y o n the departure of C u ( S a l p r n ) ; secondly, associative interchange, with a fully f o r m e d intermediate C u ( S a l p r n ) ( M b h h ( I I I ) H 2 0 ) , which then dissociates in the k3 pathway. C o m p a r i s o n o f the two-k and three-k with the four-k models ( T a b l e 4, using an F test) indicates that addition o f the fourth rate constant, k3, i m p r o v e d the data fit b e y o n d the degree e x p e c t e d f r o m simple statistical effects. U n l i k e ApoMbhh, C u ( S a l p r n ) ( A p o M b h h ) aged several w e e k s at 40( ` shows no variation in the electronic spectrum and extracts hemin. A g e d ApoMbhh ( a g e d overnight at 4°C)

,i.

.i~

~,(m)H20 1 ~ ~ 1

cq(s.llmO

J, Cu(S~mXMI,~on)HaOI Scheme 2.

J.J. Stephanos et al. / Journal of Inorganic Biochemistry 73 (1999) 137-144

143

References

...... 0

.... q . . . . . . .

I

---~

. . . . . .

~

.......

~

......

i

. . . .

Time (rain)

Fig. 7. Time dependences of the concentrations of (A) Hbc(III), (A') Cu(Salpm) (Mbhh(IlI)H20) and (C) Mbhh(Ill) (tt20) for the heme release from Hb~(llI) to Cu(Salprn) (ApoMbhh). Table 4 The k values calculated for three- and two-k models of the absorption-time curve in the reaction of Hb~ (III) and Cu (Salprn) (ApoMbhh) at 25°C Three-k model

10 3kl (min -I)

6.10

Two-k model 3.59 2.65

10-3 k_l (min -~ ) 10 4 k2 ( m i n - I ) 10-4 k3 (min ;)

6.23 1.70 1.43

2.75 1.70

3.01

F

35

2708

249

1.78 4.53 2.39

0.17 63.74 0.17 63.74

320

9170

9170

fails to extract hemin of Glycera dibranchiata hemoglobin, or Aplysia myoglobin, due to protein denaturation.

5. Conclusions

N,N'-Propylene-bis-( N-salicylidene )copper( II ) stabilizes apomyoglobin, but does not inhibit the ability of apomyoglobin to extract hemin from the main component of Glycera dibranchiata hemoglobin. The optical spectrum of this copper(II) Schiff-base complex of apomyoglobin and ESR parameters imply no change in the identity of the first coordination sphere around the copper, while hydrophobic solvatochromism cannot be excluded. The transfer of hemin from Glycera dibranchiata hemoglobin to apomyoglobin modified by copper(II) Schiff-base complex is examined. The mechanism involves dissociative and associative interchange pathways. The kinetics fit a triangular mechanism for a biphasic reaction, which involves dissociative and associative interchange pathways. Heroin replaces the copper complex. Rate constants for the transfer of hemin to copper(II) Schiff-base apomyoglobin complex and the change of concentration with time are evaluated. Missing distal His ET, the salt bridge between the propionate of hemin and the Phe ct~3, and deletion of the D-helix in Glycera dibranchiata are responsible for the destabilization of the hemin.

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