The Green Hemoproteins of Bovine Erythrocytes SPECTRAL, LIGAND-BINDING, AND ELECTROCHEMICAL PROPERTIES*

II. The two green hemoproteins isolated from bovine eryth- rocytes (form I and form II) have been characterized as to spectral, electrochemical, and chemical properties. The absorption spectra of the isolated hemoproteins are typical of high spin ferric states. Reduction of the hemoproteins yields high spin ferrohemoproteins. Complexation of the ferrohemoproteins with CO and the ferrihemoproteins with cyanide yields low spin complexes, demonstrating the presence of an exchangeable weak field ligand in both the ferrous and ferric states of the hemoproteins. The differences in position and intensity of the absorption peaks of the visible spectra allow the two forms to be distinguished from one another. The midpoint potential of forms I and II were found to be +0.075 and +0.019 V, respectively, at pH 6.4 and +0.038 and -0.005 V, respectively, at pH 7.0. This is consistent with the gaining of 1 proton/electron during the reduction. The Nernst plot reveals an unusual 0.5-electron transfer, whereas a quantitative titration demonstrates a l-electron transfer. Form I binds cyanide more tightly than form II (K,, of 84 and 252 PM, respectively). The observed spectral, and

The two green hemoproteins isolated from bovine erythrocytes (form I and form II) have been characterized as to spectral, electrochemical, and chemical properties. The absorption spectra of the isolated hemoproteins are typical of high spin ferric states. Reduction of the hemoproteins yields high spin ferrohemoproteins.
Complexation of the ferrohemoproteins with CO and the ferrihemoproteins with cyanide yields low spin complexes, demonstrating the presence of an exchangeable weak field ligand in both the ferrous and ferric states of the hemoproteins.
The differences in position and intensity of the absorption peaks of the visible spectra allow the two forms to be distinguished from one another.
The midpoint potential of forms I and II were found to be +0.075 and +0.019 V, respectively, at pH 6.4 and +0.038 and -0.005 V, respectively, at pH 7.0. This is consistent with the gaining of 1 proton/electron during the reduction. The Nernst plot reveals an unusual 0.5-electron transfer, whereas a quantitative titration demonstrates a l-electron transfer. Form I binds cyanide more tightly than form II (K,, of 84 and 252 PM, respectively).
The observed spectral, electrochemical, and ligand-binding differences between forms I and II can be explained in terms of a greater electron-withdrawing ability of the side chains of the heme of form I relative to the heme of form II. and is equal to 0.241 at pH 7.0 but is essentially zero at pH 6.4. In order to correct for the dye's contribution to the hemoprotein's absorbance at any given point in the titration, y is multiplied by the fraction of the dye reduced times the total change in absorbance at 625 nm and this product is subtracted from the change in absorbance at 444 nm.
The potentiometric data were analyzed by plotting the calculated potential versus the log of the ratio of the oxidized to reduced hemoprotein as per the Nernst equation.
The plot for the titration of form I at pH 7.0 was exactly parallel to that obtained at pH 6.4. At pH 6.4 and 7.0 the dye was not found to have an oxidationreduction behavior as would be predicted by the Nernst equation. Consequently, the dye was titrated in an anaerobic cuvette fitted with a miniature platinum electrode and reference cell and the measured values plotted to obtain a standard curve (see Fig. 1). Extrapolated values read from the curve were used to determine the potential of the protein/dye mixture at any given point in the titration.
The dye's oxidation-reduction behavior on the Nernst plot was apparently that of a l-electron acceptor when mainly in the oxidized state and that of a 2-electron acceptor when more than 50% reduced.
The electrochemical behavior of the dye at pH 7.0 was predicted from the pH 6.4 data by assuming 1 proton gained for every electron gained. The absorbance changes of the dye in the dye/ hemoprotein mixture were monitored at 625 nm, an isosbestic point of the hemoprotein.
The buffer was 0.10 M potassium phosphate, pH 7.0, and the temperature was 20°C. In Tables  I through  IV are summarized the absorption maxima and the molar absorptivities for the absolute and difference spectra of the hemoproteins in the free and liganded states. As isolated, both forms I and II possess absorption spectra with two widely separated bands in the visible region, diagnostic of high spin ferric complexes (see Refs. 5 to '7). Relative to form II, the visible bands of ferri-form I (510 to 614 nm) are less pronounced and are found at longer wavelengths.

RESULTS
Reduction with sodium dithionite under strictly anaerobic conditions converts the ferrihemoproteins to high spin ferrohemoproteins.
For each protein, reduction is accompanied by ' The abbreviations used are: form I, the first green hemoprotein to elute from DEAE-Sephadex; form II, the second green hemopro-  " (s) denotes a shoulder rather than a discrete peak.
an approximately 22 nm shift of the Soret band to longer wavelengths. Relative to form II, the shoulder in the spectrum of ferro-form I at approximately 608 nm is more pronounced. The two ferrohemoproteins react with carbon monoxide to form derivatives whose spectra are characteristic of low spin complexes, possessing sharp Soret bands near 426 nm and partially fused (Y-and p-bands. The visible bands of form I (563 and 596 nm) are of equal intensity, whereas the bands of form II are of unequal extinction, are sharper, and are found at shorter wavelengths.
A moderate 6 peak (characteristic of low spin, ferrous complexes) is detected in the spectra of the CO-ferrous complexes of both forms. The 6 peak of the CO complex of form I is at 358 nm (E,,,~ I-25) and is not quite as sharp as the 6 peak of the CO complex of form II (absorbance maximum at 339 nm, E,&, = 28). The peaks are the same in terms of extinction and position of the absorbance maximum when reduction is achieved either chemically (with sodium dithionite) or photochemically (with lumiflavin-3-acetate, EDTA, and light). The 6 peak is therefore not dependent upon the type of reductant used.
The ferric states of forms I and II react at pH 7.2 with 5 mM cyanide to form complexes with spectral features denoting low spin character (Fig. 3). The absorption peaks of the cyanide complexes of forms I and II are found at different positions and the 6 peak is more pronounced for form II.
The high spin type spectra of the ferrous and ferric states of the hemoproteins and the low spin type spectra of the cyanide . ferrihemoprotein complex and the carbon monoxide. ferrohemoprotein complex, suggest that one strong field ligand and one weak field ligand are bonded to the heme iron. The ligand exchange with the strong field ligands cyanide and carbon monoxide must take place with the weak field ligand of the protein to give the low spin spectra characteristic of complexes with two strong field ligands.
Both forms exhibit peaks in the ultraviolet region at 268 nm with slight shoulders at 261 nm. Since there is not an abnormally large amount of phenylalanine present in the proteins2 it would appear that this unusually blue-shifted absorbance maximum is due in part to the hemin absorbance. Absorbance in this region by hemoproteins has been thought to be due only in part to absorbance by the protein moiety (8).
In general, the absorption maxima in the visible region of these spectra are found at longer wavelengths for form I than for form II. This supports the conclusion, drawn from the comparison of the reduced pyridine hemochrome spectra of the hemoproteins (l), that the heme of form I has a more extended resonance pathway.
The distinctive differences between the two forms of the hemoprotein are highlighted in the difference spectra obtained by recording the reduced minus oxidized spectra, the reduced carbon monoxide minus reduced spectra, and the oxidized cyanide minus oxidized spectra (see Figs. 4 to 6).

Dissociation
Constant for Cyanide .Ferrihemoprotein Complexes -The two forms of the green hemoprotein were titrated with cyanide as described in the legend to Fig. 7. No assumption was made as to the ionic state of cyanide bound by the hemoproteins.
The spectral changes observed upon the stepwise addition of cyanide to forms I and II are shown in Plates a and b, respectively. Upon addition of cyanide, the normal  I  I  I  I  I  I  I  I  I  I  I  I  I  400  450  Ym  550  em  650 Fig. 8).
The affinity for cyanide of form I of bovine erythrocyte green hemoprotein is similar to that observed for the human erythrocyte green hemoprotein.
Thus the form I of bovine erythrocyte green hemoprotein and the human erythrocyte green hemoprotein are similar not only in terms of the reduced pyridine hemochrome spectrum (1) but also in terms of affinity / , I I , / I , , , , , I   /,,I  ,/, Fig. 9. Upon successive additions of dithionite, the absorbance of the Soret band at 416.5 nm was seen to decrease with a concomitant increase in absorbance of the Soret of the reduced form at 439 nm, and the visible peak at 573 nm. Four sharp isosbestic points were observed, showing that a single spectral species was converted to another single species. The plot of these results (Fig. 9 Fig. 3. In the figure the ordinate scale at the right is a A absorbance scale which applies to wavelengths longer than 485 nm and that on the left to wavelengths shorter than 485 nm. consumed for every molecule of hemoprotein converted to its reduced form. Since dithionite is a 2-electron reducing agent, it is evident that 0.86 electron was taken up for each molecule of hemin reduced. The anaerobic titration of form II also revealed the transfer of 1 electron/atom of iron (data not shown). The spectral changes observed were parallel to those observed for form I. Analysis of data as described for form I demonstrated that 0.54 molecule of sodium dithionite was consumed/atom of iron. Thus, 1.08 electrons were consumed in the reduction of each iron atom. The accuracy of these reductive titrations appeared to be approximately &lo%. The potentiometric behavior of the two hemoproteins during titration with dithionite in the presence of the indigotetrasulfonate at pH 6.4 is shown in Fig. 10. The data plotted according to the Nernst equation should lie on a straight line if a single species is present. The biphasic behavior of form I is clearly indicative of two species, in an approximate ratio of 4:l. The minor species (of lower, more negative potential) has a behavior identical to form II and is due to contamination of this preparation of form I by form II. The potentiometric titration of form II shows one species to be present. The midpoint potentials for forms I and II at pH 6.4 are +0.075 and +0.019 V, respectively. At pH 7.0, the midpoint potentials of form I and form II are +0.038 V and -0.005 V, respectively. The standard deviation of these readings is approximately The experimentally determined values of -0.062 V/pH unit for form I and -0.070 for form II are in strong agreement with a mechanism which includes the addition of 1 proton for every electron transferred to the oxidized hemoprotein.
Theoretically, the transfer of 1 proton/electron results in a potential change of -0.059 V/pH unit.
From the slopes of the lines in Fig. 10 (0.118 V/log(oxlred)) the number of electrons involved in the reduction of forms I and II in the presence of indigotetrasulfonate was calculated to be 0.5. A value of n = 1 would have been predicted for a lelectron transfer of Fe+:s + 1 e-' + Fe=. This evidence for a one-half electron transfer indicates that the mechanism of electron uptake by the hemoprotein does not occur on a simple 1 electromheme basis. However, the stoichiometric titration of both forms I and II with a standardized solution of sodium dithionite did give the expected value of near unity.
Several discrepancies of this sort have been reported. Guengerich et al. (12), for example, observed a 2-electron stoichiometry in the reduction of cytochrome P-450, whereas a value of n = 1 was deduced from potentiometric data. This anomaly has been resolved, the stoichiometry of reduction now being unity (13). A possible explanation for the rz = 0.5 value for the green hemoprotein would be site-site interactions.
Since the hemoproteins in solution exist as monomers with only 1 hemel monomer, this postulate would necessitate transient aggregation of the hemoprotein.
No spectral evidence for such aggregation has been observed in either the chemical or photochemical reductions. Correlation of Spectral, Electrochemical, and Ligand-binding Properties -The spectral, electrochemical, and ligandbinding differences between forms I and II can be explained in terms of a greater electron withdrawing ability of the side chains of the heme of form I relative to the heme of form II. Evidence for such a difference in the two hemes was provided by the observation that the pyridine hemochrome spectrum of form I is red-shifted relative to form II (1). Pyridine hemochrome spectra provide direct information concerning the electron withdrawing ability of hemes since the spectra are insensitive to the protein moiety under the protein-denaturing conditions employed. Moreover, the pyridine hemochrome spectra of the protein-free hemes showed the same differences. 4 As predicted for a more conjugated heme, the visible absorbance maxima of the ferrous, CO-ferrous, ferric, and CN--ferric species of native form I are found at longer wavelengths than the corresponding maxima of form II; analogous spectral differences are observed when various hemes with known electron-withdrawing capacities are inserted into apo-myoglobin (14,15). The greater affinity of form I for cyanide is likewise consistent with a more highly conjugated heme; increasing the electron-withdrawing ability of hemes in hemoproteins is known to result in stronger binding of sigma (o-1 donor ligands such as cyanide (14,16). The more positive oxidation-reduction potential of form I relative to form II (+0.038 V uersus -0.005 V) can also be explained in terms of structural differences in the hemes; the presence of electronwithdrawing groups on hemes is known to make the oxidationreduction potential more positive (17). If there actually are differences between the apoprotein moieties of the two forms, then they are of a subtle enough nature as not to reverse the  Fig. 7. It was assumed that both hemoproteins were completely converted to the cyanide trend in electrochemical and ligand-binding properties as predicted by analysis of the spectrum of the reduced pyridine hemochrome.
The capacity of the green hemoproteins to exchange ligands and the observed rapid rate of autoxidation of the ferrous for-n? are compatible with the idea that these hemoproteins function as oxidases. Preliminary studies have shown that the hemoproteins can be reduced by solubilized liver microsomal electron transport systems." However, if they do indeed function as oxidases, the substrates which could provide electrons for the hemoproteins in the erythrocyte remain unknown.