ON THE METABOLIC BREAKDOWN OF HEMOGLOBIN AND THE ELECTRONIC STRUCTURE OF THE BILE PIGMENTS BY BERNARD PULLMAN AND ANNE-MARIE PERAULT*

INSTITUTE FOR MUSCLE RESEARCH AT THE MARINE BIOLOGICAL LABORATORY, WOODS HOLE, MASSACHUSETTS Communicated by Albert Szent-Gyorgyi, August 24, 1959 The metabolic breakdown of hemoglobin consists, in its first phase, in an oxidative cleavage of the protoporphyrin ring, through the elimination of the methine group situated between the two pyrrole rings carrying the vinyl substituents (a-methine group). The product formed, choleglobin, is then easily split into globin, ferric ions and a green pigment, biliverdin (I) which is an open chain conjugated totrapyrrole. The further metabolic transformations consist in a chain of reductions: biliverdin OH2C3H502 C3H602 0H2CH2 C3H502 C3HSO2 CH2 CH3 CH COH3 CH3 COH3 C-H OH CH COH CH3 CkLOH )C tLCH CH=A LCH' H2 OCH) HON NH N NOH HO N NH NH N OH (I) Biliverdin (II) Bilirubin is reduced to bilirubin (II), a pigment of orange color and possibly also to a dark brown pigment, called urobilin (III), through the conversion of, respectively, the central or the two terminal methine groups, -CH=-, to methylene groups -CH2-. Further reductions lead through the hydrogenation of all the methine and the vinyl groups to urobilinogen, and through the complementary partial hy- drogenation of the two terminal pyrrole rings to stercobilin. C3H502 C3H502 CH,3 C2HOCH3,H3CKH ,C2H5 )J~Il-OH2-, -CH==Q CH2,K\ HO NH NH N NHOH (III) Urobilin Biliverdin and bilirubin are the principal bile pigments. This process of metabolic degradation through successive hydrogenations is to some extent peculiar as the general scheme for the metabolic degradation of conjugated biological substances, e.g., purines and pyrimidines of the nucleic acids, consists rather in an extensive series of successive oxidations, followed eventually by hydrol- ysis. The present study was undertaken in view of finding an interpretation for this state of affairs. The Calculations.-The calculations have been carried out by the molecular or- bital method in the L.C.A.O. approximation.2 They refer to the electronic prop- erties of the 7r electrons pool of the compounds. The saturated groups present in the molecule have been considered to have only a negligible influence on these prop- erties and have been omitted from calculations. Protoporphyrin has thus been assumed to be simply a 1,4-divinyl porphyrine (IV) and biliverdin has been repre- sented by the simplified formula (V). Bilirubin has been considered to be composed of two separated conjugated fragments VI and VII, which are both substituted 1476 Downloaded by guest on September 26, 2021 VOL. 45, 1959 BIOCHEMISTRY: PULLMAN AND PERAULT 1477

dipyrroles. Dipyrrole VIII represents also the central conjugated fragment of urobilin. 4CH2 OH2 CH2 CH~~~~~~~~~~~~~~~~~~~III

OH HCCHH OH NHILCH4(¢3 [ULC NH

(IV) (V) OH2 OH2

HO N NH NH N OH NH N (VI) (VII) (VIII) A unique value atN = ac + 0.7,3, has been attributed to the coulomb integral of the four nitrogen atoms of the compounds studied, this value being intermediate between the one that we adopt usually3 '4for a pyridine type nitrogen (aN = a, + 0.4o,,) and a pyrrole type nitrogen (aNH = a, + #,,)- Previous studies, by a series of different authors5 on properties of the unsubstituted porphyrine ring show effectively that the changes introduced by adopting different values of this param- eter for the hydrogen-carrying and hydrogen-free central nitrogens or by consider- ing the different possible tautomeric forms are rather small and irrelevant for the description of the fundamental electronic characteristics of this ring, such as the values of the electronic energy levels or the distribution of the electrical charges. These two characteristics will, in fact, be those which we shall essentially take into consideration in this paper. May we remind the reader3 , 4that in the L.C.A.O. molecular orbital method the energies of the molecular orbitals of the mobile or Tr electrons of the system are expressed in the form Ei = a + Kid, where a is the coulomb integral and A the resonance integral of the method. The energies of the individual orbitals are thus characterized essentially by the value of the correspond- ing Ki. Positive values of Ki correspond generally to orbitals occupied in the ground state of the molecule (bonding orbitals) and negative values of Ki to orbitals empty in that state (antibonding orbitals). The smallest positive value of Ki cor- responds to the highest occupied molecular orbital (h.o.m.o) and the smaller this value the greater the electron-donor capacity of the molecule. The smallest nega- tive value of Ki corresponds to the lowest empty molecular orbitals (l.e.m.o) and the smaller this value the greater the electron-acceptor capacity of the molecule. Results and Discussion.-The table gives the energies of the highest filled and low- est empty molecular orbitals and the figure the distribution of the electrical charges (in electronic units) in the compounds studied. TABLE 1 Energy of the Energy of the Highest Occupied Lowest Empty Compound Molecular Orbital Molecular Orbital Protoporphyrine IV 0,293 -0,233 Biliverdin V 0,455 0,021 Vinyl-hydroxydipyrrole VI 0,468 -0,251 Dipyrrole VIII 0,618 -0,252 Downloaded by guest on September 26, 2021 1478 BIOCHEMISTRY: PULLMAN AND PERAULT PROC. N. A. S.

The principal conclusions which may be drawn from these data seem to be the following: (1) Protoporphyrin constitutes at the same time a good electron donor and a good electron acceptor. This property is, in fact, a general characteristic of porphyrins and is, most probably, of primary significance for their biological functioning. In connection with our previous study on the structure and functioning of the coen- zymes of oxido-reduction4 it appears particularly plausible that the electron donor and acceptor properties of the porphyrins are of importance in determining, in con- junction with the similar properties of the central metal atom, the role of the cyto- chromes as electron carriers. (2) The oxydative metabolic rupture of the hemoglobin occurs at the electron richest methine bridge. In fact, all the methine carbons of the porphyrine ring are rather slightly electron deficient, bearing less than the one X electron which they contribute to the electron pool. They carry thus, in fact, a small positive charge (which may be obtained by subtracting from unity their electronic charge). The metabolic rupture of the macro-ring occurs thus at the less positive methine carbon. CH2 1.010 ~CHo.994 1.055 41.02 1.lob CH2 \ 0.964§ 0.982 ,0.978 0.958 0.994CH I 1.032 0.979 1.480 .988 1.049 N1.480 1.487 N 1.050 6.982 1486 0.988 1.046

1.04 1.046 Protoporphyrin (IV) 0.971 H2 1.04,; H2

CH 0.993 0.986 H 1.134 .993 1.052 1.031 1.036 1.053 1.026 .085

0.863 OH824 0.998 CH 0.995 H 1.008 0.838 )t> C H'Po.76CH CHC-;0.760 0.13 3 HO N N N N OH 1.904 1.474 1.402 1.393 1.468 1.896 Biliverdin (V) 1.00 LTH2

0.994 CH 1.152 1.021 1.070 1.125 1.118 1.049 1.049 1.118

0.904 1 H 1.064 1.000 0.980 1.059 1.059 0.980 0.814 0.756 HO N N N N 1.914 1.469 1.422 1.416 1.416 Dipyrrole (VI) Dipyrrole (VIII) FIG. 1-Distribution of electronic charges. Downloaded by guest on September 26, 2021 VOL. 45, 1959 BIOCHEMISTRY: PULLMAN AND PERAULT 1479

(3) The rupture of the porphyrine ring and the formation of an opened tetra- pyrrole are accompanied by a drastic redistribution of the electronic energy levels and in particular of those corresponding to the highest occupied and lowest empty molecular orbitals. Thus, biliverdin seems to possess a very unusual property, which has not yet been observed in another compound, namely, that its lowest empty molecular orbital is a bonding one (K = 0.021), the sign of its coefficient being that generally associated with orbitals which are already occupied in the ground state of molecules. This signifies that this molecule must possess unusually pronounced electron ac- ceptor properties. It will be eager to fill up all its bonding orbitals and will conse- quently be particularly easily reductible. This characteristic may then be considered as responsible for the apparently uncommon orientation imposed on the mechanism of metabolic transformation of hemoglobin.t The possibility thus shown of the existence of compounds possessing the unusual characteristic of a bonding lowest empty orbital is complementary to the possibility which we have recently shown4'6 of the existence of compounds in which the highest filled molecular orbital is an anti- bonding one. (4) The reduction of biliverdin occurs essentially at the central methine carbon and only to a much lesser extent at the terminal ones. In connection with this, it may be remarked that the central methine carbon is particularly greatly electron deficient and will consequently have a strong tendency to complete its electronic gap. Energy considerations also favor the reduction at the central methine bridge: the loss of resonance energy corresponding to this reduction is practically negligible (0.053,-- 1 Kcal/mole), while it is of the order of 0.43( 8 Kcal/mole) for the double reduction on the two terminal methine carbons. t (5) Both in bilirubin and in urobilin there still is a conjugated dipyrrole system which will undergo a further reduction. It may be noted that this dipyrrole, whether substituted. as in bilirubin, or unsubstituted, as in urobilin, does not possess any more the unusual property of a bonding lowest empty orbital. Nevertheless, the lowest empty orbital of these dipyrroles, although antibonding, is still a very low- lying one, which means that these systems will still be excellent electron acceptors and, consequently, should be, as they apparently are, easily reductible. (We have calculated the energies only for the vinyl-hydroxy-dipyrrole VI, but there is no doubt that the properties of the isomeric vinyl-hydroxy-dipyrrole VII will be very similar.) The unusual property of possessing a bonding lowest empty molecular orbital appears to be de- pendent on the number of pyrrole rings in the polypyrrole chain: it is not present in the dipyr- role chain but manifests itself in the tetrapyrrole chain. (In the unsubstituted tetrapyrrole analog of biliverdin the bonding character of this orbital should be even more pronounced than in biliverdin itself: its K, = 0.064.) We are investigating, at present, the general conditions necessary for the appearance of this unusual property. This work was sponsored by a grant (C-3073) of the United States Public Health Service. It was prepared while one of the authors (B. P.) was attending the Con- ference on Submolecular Biology at the Institute for Muscle Research at Woods Hole. This author wishes to thank Dr. Albert Szent-Gyorgyi for useful discussions. * Permanent address: Institut de Biologie Physico-Chimique, Universit6 de Paris, 13 rue Pierre Curie, Paris 5e, France. t It may be reminded that the purines and pyrimidines of nucleic acids, which are metabolically degraded by a series of successive oxidations. are electron donors.3 Downloaded by guest on September 26, 2021 1480 XiOCHEAMIS7'RY: N. SUEOKA PROC. N. A. S.

t Porphyrins are endowed, in general, with a very great resonance energy which is responsible for the relative stability of these molecules. Thus, the resonance energy of the protoporphyrine IV is 10,570 ,3, which, with the usual value adopted for i3 in this type of calculation, namely B = 20 kcal/mole, represents about 210 kcal/mole. Although the rupture of the cyclic macro-ring represents a loss of a part of the resonance energy, this energy is still appreciable in the bile pig- ments: e.g., the resonance energy of biliverdin is 9,851 ,3 which is nearly 200 kcal/mole. l For more details about these transformations see, e.g., R. Lemberg and J. W. Legge, "Haema- tin Compounds and Bile Pigments" (New York: Interscience Publishers, 1949), or C. H. Gray, "The Bile Pigments" (London: Methuen Ltd., 1953). 2 For a general description of the method see, e.g., B. Pullman and A. Pullman. "Le theories 6lectronique de la chimie organique" (Paris: Masson Ed., 1952). 3 Pullman, B., and A. Pullman, these PROCEEDINGS, 44, 1197 (1958). 4 Pullman, B., and A. Pullman, these PROCEEDINGS, 45, 136 (1959). 5 E.g., H. C. Longuet-Higgins, C. Rector, and J. R. Platt, J. Chem. Phys. 18, 1174 (1959); T. Nakajima and H. Kon, J. Chem. Phys. 20, 750 (1952); S. L. Matlow, J. Chem. Phys. 23, 673 (1955); G. R. Seely, J. Chem. Phys. 27, 125 (1957); H. Kobayashi, J. Chem. Phys. 30, 1373 (1959). 6 Pullman, B., and A. Pullman, Biochim. et Biophys. Acta (in press). See also G. Karreman, I Isenberg, and A. Szent-Gyorgyi, Science (in press).

A STA TISTICA L ANALYSIS OF DEOXYRIBONUCLEIC ACID DISTRIBUTION IN DENSITY GRADIENT CENTRIFUGATION BY NOBORU SUEOKA THE BIOLOGICAL LABORATORIES, HARVARD UNIVERSITY Communicated by Paul Doty, August 2.5, 1959 Meselson, Stahl, and Vinograd (1957)1 have established a powerful technique for providing information on molecular weight, density, and their heterogeneities in one operation. A brief description of the way the technique is applied to deoxy- ribonucleic acid (DNA) is as follows; two to three micrograms of DNA in a 7.7 molal cesiunm chloride solution of density 1.7 are centrifuged in the SPINCO Model E analytical ultracentrifuge. After an equilibrated density gradient of cesium chloride is established, DNA molecules converge to a position in the gradient cor- responding to its density and form a narrow band. Theoretically it has been shown that in the absence of heterogeneities of both density and molecular weight the distribution of DNA molecules is Gaussian with a standard deviation (a-) which is a function of the molecular weight of the DNA sample used. In the presence of molecular weight heterogeneity, both number and weight average molecular weights can be derived from the band profile. I A DNA. sample can have, however, both molecular weight and density hetero- geneities. Recently it has been shown that the molecular density of DNA is re- lated to its base composition.23 It has been also shown that heat denaturation4 2 and incorporation of either 5-bromouracil,I or heavy isotopes of nitrogen44 and carbon6 change the density. In order to analyze the experimental distribution of DNA in the band when both molecular weight and density heterogeneities exist, a general theory is pre- sented and its applications to sonicated molecules of calf thymus and pneumococcus DNA are described. Downloaded by guest on September 26, 2021