N -B R o M o s u c c I N I M I D E - c Y T o c H R o M E
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Spectroscopic Properties of N-Bromosuccinimide-Modified Horse Heart Cytochrome ct Yash P. Myer* and Pranab K. Pal
ABSTRACT : The N-bromosuccinimide-modified horse heart cytochromes c in the oxidized state of heme iron in the p H range 2-10 exhibit four different spectroscopic types. In the acid medium (type I) as well as in the alkaline medium (type IV), the spectral characteristics of the modified preparations are very similar to those exhibited by the native protein under identical conditions. At intermediate pH’s (pH 4-5, type 11; pH 6-8 type III), there are two distinct molecular forms, the relative proportions of which change with increasing modification of the protein. The proportion of the first form, which exhibits low-spin characteristics over the entire pH region 3-9 much like those of the native protein, reaches a maximum value in the preparation at a 3: 1 NBS/protein ratio, while the proportion of form 11, which exhibits a high-spin spectrum a t pH’s 4-5 (type 11) and changes to a low-spin form lacking
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the 695-nm band with a pK of about 5.4, increases monotonically. A similar two-molecular-form situation is also present in the spectra of the modified preparations in the reduced state of iron. The extent and the nature of alteration of functional groups in various modified preparations, and their relative NADH-reductase and succinate activities, have been related to the coordinational configuration of the two forms. Form I, containing modified tryptophanyl residue 59 and methionyl residue 65, exhibits a spectral behavior and succinate oxidase activity very similar to that of the native protein, but the NADH-reductase activity is deranged; form I1 is the molecular species produced in a consecutive manner by additional modification of methionyl residue 80; the coordination configuration of the heme iron is altered, which results in the loss of the succinate oxidase activity of the protein as well.
he elucidation of structure-biological function relationships in proteins through studies of effects of chemical modification of amino acid functional groups, of necessity, requires investigation of multiple facets : first, the structural localization of the chemical modification and studies of its effects o n the biological function of the molecule; second, the structural interpretation of the reactivity of the various functional groups; and third, the implications of the chemical modification upon the conformation of the molecule. In the case of the reaction of horse heart ferricytochrome c with N bromosuccinimide (NBS), in the preceding two papers we have dealt primarily with these aspects of the investigations. However, in the system under question, cytochrome c, one important aspect-the effect of the chemical modification o n the nature of the central-coordinated complex of the heme iron-needs further consideration, since it constitutes the seat of biological function, the electron-capture and the electron-donor properties in the electron-transport chain. In this article, we have been concerned with this facet of the structurefunction relationships in this molecule.
from amino acid analysis and the relative N A D H cytochrome c reductase and succinate oxidase activities are listed in Table I for each preparation used in these investigations. Spectroscopic measurements were conducted on a Cary 15 spectrophotometer, and the p H measurements were made using a type B glass electrode and a Radiometer Model 26 pH meter. The titrations were conducted in a specially designed apparatus, the details of which are reported in a recent article (Myer and Harbury, 1972). The solutions during these investigations were maintained constantly under a n inert atmosphere of ultrapure helium, and the addition of the reagent, acid or base, was made by ultraprecision syringes. The reduction of the solutions was performed by direct addition of solid dithionite t o the apparatus, while the pH of the solution was continuously monitored and maintained automatically at a prescribed value with the aid of a Radiometer autotitrator. The concentrations of the solutions were determined spectrophotometrically using the appropriate extinctions at 506 nm, the isodichroic point for the NBS reaction (Myer, 1972a).
Materials and Methods
Results
Native horse heart cytochrome c , type 111, was Purchased from Sigma Chemical Co. ; the NBS derivatives used for these investigations are those whose preparation and structural, functional, and conformational characteristics are reported in the preceding two articles (Myer, 1972ah). The results
The 3 NBS-modified ferricytochrome c between pH 2.0 and 10.0 exhibits four spectroscopically distinguishable types (Figure 1). Type I, a stable molecular form below pH 2, converts to type 11 with an apparent p~ of about 3.0, which subsequently changes t o type 111with a pKof about 5.4. The stable type at neutral pH finally changes t o type IV with a mid-pH of about 9.5. The four types exhibit pH regions Of p H 2.0,4-5,6-8, and at alkaline pH’s, respectively. The visible spectra of the 3 : l NBS-modified preparation in the ferric state of heme iron corresponding to the four types are shown in Figure 2, and in Figures 3 and 4 the absorption curves for both the ferric and ferrous forms of each preparation are compared. The positions of the spectral bands are listed in
From the Department of Chemistry, s t a t e University of N~~ y o r k at Albany, Albany, New York 12222. ReceiuedJune 15, 1972. This work was supported by Research Grants 68-13524 and GB-30687X from the National Science Foundation. This article constitutes paper VI11 of the series entitled : Conformation of Cytochromes. A preliminary report of this work was presented at the Biophysical Society meeting, Los Angeles, 1971. 1 Abbreviation used is: NBS, N-bromosuccinimide.
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TABLE I : Amino Acid Content and Relative Activities of Various NBS Cytochromes c .
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FIGURE 1 : Spectroscopic titration
of native and NBS-cytochromes c in acidic and basic pH ranges. (A) Acidic pH range; (B) basic pH range. In a part A, filled markings designate the titration from pH 7 to 1, and unfilled markings, the reverse titration. ( 0 , O ) Native; (A, A) 3 : l NBS-modified preparation; ( 3 , m) 6 : l NBS-modified preparation.
Table I1 for the ferric 3 :1 NBS-modified preparation along with those of the native protein for ease of comparison. Type I, observable in extremely acidic conditions, exhibits characteristics similar to those of the native protein under similar conditions. The position of the Soret maximum at 394 nm, a visible maximum at 495 nm with a well-defined shoulder at 530 nm, and the well-resolved 618-nm peak are all spectral features typical of the high-spin form of the hemoprotein (Day et ul., 1967) and simple heme-peptide systems (Myer and Harbury, 1972). Type I1 does not appear to have a n analog in the spectroscopic behavior of the native protein, and it exhibits rather peculiar spectroscopic characteristics (Table I1 and Figures 2 and 3). It contains a peak at 528 nm with a well-defined shoulder at about 565 nm, and a visible minimum a t 494 nm, features similar to those of the native protein (Table II), which is characteristic of the low-spin state of heme iron, on the one hand, and on the other, it has a well-resolved peak at about 615 nm (Figure 2), which is characteristic of the high-spin form of the protein. In addition, Type I1 exhibits a well-defined peak in the 690- to 700-nm region, a spectral feature attributed to the presence of methionyl sulfur coordination to heme iron (Schechter and Saludjian, 1967) and also to the low-spin state of the metal atom (Zerner et ul., 1966). The position of the Soret band, 403 nm as compared to 409 nm for the native protein at this pH, is also indicative of the complexity of the spectrum. The transformation of type 11 to type 111 with a pK = 5.4 results in changes which eliminate the spectral pecularities of type 11. The low-spin characteristics become more pronounced---i.r., the 528-nm peak is enhanced and the Soret band is moved to 407 nm-with concurrent elimination of the high-spin band at 615 nm (Figures 2 and 3; Table 11). The spectrum in the region 690-700 nm is more or less unaltered, except for a small decrease of extinction over the entire region (Figure 2). Type IV retains essentially all the features of type 111, except that the Soret band is shifted to the blue with enhanced absorptivity, and there is a slight lowering of extinction in the visible bands; type 1V is devoid of any known characteristics of the high-spin state of heme iron. The most significant change is the elimination of the peak in the 690- to 700-nm region. The spectral changes associated with the transformation of types 111-IV are similar to those observed for the native protein undergoing transition in the same pH range (Table 11): and the spectral characteristics are typical of the low-spin form of cytochrome c lacking the criteria for the presence of
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Prepn (NBS/ 0 . 0 0 3.01 6 02 9 00 Protein, MoleslMole): (Native) Amino Acid Content (Moles/Mole of Protein) Tryptophan 1 . 0 0 . 0 2 0.00 0.00 Methionine sulfoxide" 0.0 1. 3 1 .6 1.8 Methionine ' 2 0 0,7 0.4 0.2 Tyrosine' 4 . 0 3.3 2.7 2.1 Histidine 3.0 3 . 0 2.9 2.9 Activity NADH-cytochrome c 100 28 reductase activity (ZAODSso/minper pmole)d Succinate oxidase 100 68 activity rate at 6 p)vi concentration)d
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