Oxidase and Other Hemeproteins - ACS Publications

12 Structure-Function Relationships in Cytochrome c Oxidase and

Downloaded by UNIV OF GUELPH on January 22, 2018 | http://pubs.acs.org Publication Date: June 1, 1971 | doi: 10.1021/ba-1971-0100.ch012

Other Hemeproteins WINSLOW S. CAUGHEY Arizona State University, Tempe, Ariz. 85281 Changes in porphyrin structure, axial ligand, oxidation state, and medium result in significant effects upon a number of independently observable physical parameters which may serve to establish in much greater detail those aspects of structure which determine hemeprotein function. Infrared spectroscopy has become a useful probe of ligand binding in hemoglobins, myoglobins, and cytochrome c oxidase. The highly characteristic properties (e.g., NMR, EPR, IR, and electronic spectra) of μ-oxohemin dimers made possible recognition that hemin a of the oxidase can form a μ-οxο dimer despite the presence of the bulky 1-hydroxy-2(trans, trans-farnesyl)ethyl group—a group which can assume a conformation in which the external double bonds could couple with iron porphyrin in electron-transfer or phosphorylation functions.

/ ^ u r recent studies on the chemistry of cytochrome oxidase function are discussed after briefly considering the value of certain physical observables for the study of hemeproteins. The porphyrin proteins and enzymes have an intriguing bioinorganic chemistry, not only because of their critical biological roles but also owing to the challenge of being able to correlate a large number of inde pendent observable physical parameters with those aspects of structure which determine biological functions. It is particularly significant that these physical observables frequently can be applied to intact tissues as well as to isolated protein and also that pure hemes and hemins can be used i n the refinement of interpretations of the effect of structure upon these parameters. It is frequently most important that the native natural 248 Dessy et al.; Bioinorganic Chemistry Advances in Chemistry; American Chemical Society: Washington, DC, 1971.


12.

CAUGHEY

Cytochrome c Oxidase

249

hemes be used if such refinements are to be significantly relevant to the in vivo environment. This point w i l l become particularly clear when we consider unique aspects of heme A structure. The significant variables found among hemeproteins obviously i n clude porphyrin structure, axial ligand, oxidation state, and medium provided by protein and/or solvent at the heme site ( J ) . Changes i n axial ligand affect the strength of binding interactions between iron and porphyrin nitrogens; conversely, changes i n groups at the periphery of the porphyrin ring alter i n turn the basicity of porphyrin nitrogens, the bonding between porphyrin nitrogens and iron, and the bonding between iron and the axial ligands. Such cis and trans effects have been well documented for iron (II) porphyrins and to a lesser extent for hemeproteins ( J , 2, 3, 4 ) . Recently, more detailed interpretations of such effects in iron (III) compounds have been obtained ( 2 ) .

Figure I. Deuteroporphyrin IX dimethyl ester iron(III) (deuteronemin dimethyl ester) The magnitude of changes i n several physical properties as the axial ligand is varied has been shown for a series of deuterohemins (Figure 1). " X " represents the axial ligand varied i n these high-spin iron (III) compounds. Rather marked effects of ligands upon the near infrared band, the quadrupole splitting of Mossbauer spectra, and zero-field splitting measured directly from far infrared spectra are illustrated by data i n

Dessy et al.; Bioinorganic Chemistry Advances in Chemistry; American Chemical Society: Washington, DC, 1971.

250

BIOINORGANIC CHEMISTRY

Table I.

Spectroscopic Properties of Deuteroporphyrin IX Dimethyl Ester Iron (III) Compounds NIR Band max. (CHCk), my.


Ligand

AE, 298°K, Mm/Sec

4.2°K Cm~

l

FciBri-

800 910 934 958

0.59 0.91 1.12 1.30

11.117.9 23.6 33.0

CHsO*oCH COONa~

761 769 836 868

0.54

9.3 10.9 13.8 14.8

3

400

500

0.6 0.7

600 700 900 WAVELENGTH -ηψ

1100

Figure 2. Absorption spectra for deu teroporphyrin IX dimethyl ester iron(III) derivatives in chloroform; ligands correspond to "X" of Figure 1 Table I (2,5,6,7). Figure 2 further illustrates changes i n electronic spec tra for chloroform solutions. I n benzene, electronic spectra, though slightly different, are generally similar (Figure 3 ) . Such differences caused by solvent effects may not be unlike subtle differences found between certain hemeproteins. The shifts i n electronic spectra attributed to changes i n axial ligand follow the same ligand order as that for the other physical



12.

CAUGHEY

251


400

900

COO 700 900 WAVELENGTH-n> SOLVENT BENZENE

1100

Figure 3. Absorption spectra for deuteroporphyrin IX dimethyl ester iron(III) derivatives in benzene. 2D values refer to zero-field splitting (7). Ligands correspond to "X" of Figure 1. properties including E S R spectra which show significant differences i n the width of the g = 6 band and N M R spectra where magnitudes of paramagnetic shifts vary ( 5 ) . Proton N M R spectra for azido and phenoxo derivatives i n C D C 1 are shown i n Figure 4. Particularly noteworthy are the large paramagnetic shifts not only for porphyrin protons but also for protons on the phenoxo ligand. Assignments are not difficult to make with such hemins and provide a basis for the application of N M R to the study of paramagnetic hemeproteins. However, assignments i n the case of the proteins are more difficult; separation of contact, pseudo-contact, and ring current field effects represent a major problem. The magnitude of ring current field effects is illustrated by the influence of concentration upon the pattern of ring methyl and ester methyl resonances for the diamagnetic 2,4-dipropionyldeuteroporphyrin I X dimethyl ester i n C D C 1 (Figure 5 ) . As the concentration is increased, an equilibrium between monomeric and dimeric species is shifted i n favor of more dimer ( 8 ) . Plots of chemical shift vs. concentration (Figure 6) reveal the 5-methyl group resonance as less sensitive to dimer formation than are 1, 3, and 8 methyls—a reflection of the manner i n which the dimer is formed. 3

3


252


X

οI

AZIDOHEMIN


iO X

ο ο ο ο CVJ X ι

. TMS

r

100

50

-50

-100

ΙΟ

δ

PHENOXO

S

Id X

" l CM

-100

50

-50

100

CHEMICAL SHIFT IN PPM

Figure 4. Proton magnetic resonance spectra at 100 MHz of azido (upper) and phenoxo (lower) derivatives of deuteroporphyrin IX di methyl ester iron(lII) in CDCl at 35°C s

Shifts i n frequency can occur as a result of intermolecular ring current field effects present in the dimer but not i n the monomer (8, 9, 10, 11). The other spectroscopic techniques mentioned above have also been applied to hemeproteins. T h e far infrared determination of zero field splitting has been extended to myoglobin fluoride (11.9 cm" ) and hemo1


12.

CAUGHEY


253

globin fluoride ( 12.5 c m ' ) and compared with protohemin fluoride ( 10.0 cm" ) and deuterohemin fluoride (11.1 cm" ) (12). Also, a nonzero Ε value (E/D ca. 0.085) was found for protohemin azide i n support of a contribution of the azide ligand to rhombic distortion i n the absence of either the protein or a irans-histidine, as is the case for myoglobin and hemoglobin azides. 1


1

1

'

40

'

'

30

'

»

40

'

ι

I

I

I

30 40 30 CHEMICAL SHIFT (8)

I

40

I

I

30

40

ι

ι

30

Figure 5. Proton magnetic resonance spectra at 60 MHz of 2,4-dipropionyldeuteroporphyrin IX dimethyl ester at different concentrations in CDCl at 35°C. Arrows indicate ester methyls. 3

NH PROTONS

RING METHYL PROTONS

_

3

5

1

aο

Λ δ.

ί \ /

_

8

0

II CCH CH 2

I I / / ® / I / / 3~^ CH

—

40

Ij// 1 1 / ® ^ 7!

Γ

tl

CH

l// 36

®

ϊ

3

HN>-NH

3

Η 3

/

ο II

VCCH2CH3 \

N-Λ

®

/

© / " /

°/

/

"

P

f ft f _L/

Ο /

I

1

-40

32

-50

CHEMICAL SHIFT (8)

Figure 6. Plots of chemical shifts vs. concentration for ring methyl and NH protons from 60 MHz proton mag netic resonance spectra of 2,4-aipropionyldeuteroporphyrin IX dimethyl ester in CDCl at 35°C 3



254


While it is clear that these approaches along with x-ray structure determinations represent highly promising probes of hemeprotein structure, one can nevertheless be severely limited in his interpretation of data in terms necessary to explain enzyme function. The identity of the axial ligands bound to metal in a given hemeprotein under a given set of conditions is frequently unclear. Furthermore, even if ligands are known, one cannot necessarily assume knowledge of the stereochemistry, strength of binding, and protein or solvent environment of the ligands—each a factor of importance to the properties exhibited by the hemeprotein. If the ligand contains protons, N M R spectra can be useful. More recently, we have explored the use of infrared techniques i n this regard. Infrared spectra of aqueous solutions of hemoglobins and myoglobins exhibit a "window" of relatively low absorption from about 1750 to 2800 cm" . Bands for carbon monoxide, azide, and cyanide ligands bound to these hemeproteins have been studied by an infrared difference technique developed by Alben and M c C o y in our laboratory. The C O band was readily observed bound to hemoglobin within red blood cells (13). The azide derivatives for each protein exhibited two bands ( F i g ure 7) assigned to low- and high-spin forms (14). W i t h myoglobin, two C O bands are observed (Figure 8) (15), whereas only one C O band was found for normal hemoglobins (13). Thus, with both C O and N derivatives, myoglobin shows a greater tendency to bind ligands in two 1

3

Figure 7. Infrared difference spectra. Top: of azidometmyoglobin vs. metmyoglobin (0.02M). Middle: of azidomethemoglobin vs. bovine plasma albumin (0.01M). Bottom: of sodium azide in 0.05M citrate buffer (pH 3) vs. buffer.


12.

CAUGHEY

255


Jl


Mb-CO (PROTOHEME)

1

JUl WATER VAPOR

Mb-CO (DEUTEROHEME) j -

ι Ii ιΐιίιιι JtftàiÉilii

..

&

AVÈÉÉÈÊÈÊ

J J u Mb-CO (MESOHEME) ι "Ï95Ô

1900

FREQUENCY (cm ) 1

Figure 8. Infrared difference spectra of carbonyl sperm whale myoglobins reconstituted from different hemes vs. metmyoglobin in phosphate buffer, pH 6.4. Top: protoheme. Middle: deuteroheme. Bottom: mesoheme. ways than does hemoglobin. W e conclude from infrared data that both C O and N form significantly bent F e - l i g a n d bonds i n hemoglobins and myoglobins (Figure 9) (16). Bending appears greater i n the case of myoglobins since vco is

Oxidase and Other Hemeproteins - ACS Publications

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