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The binding of carbon monoxide and weak or strong field ligands to deuteroheme has been studied over the widest possible ranges of carbon monoxide and ligand concentrations. The variations of the affinity constants of the monoliganded hemes for carbon monoxide (and of monocarbonylheme for a sixth ligand) have been related to the relative a-donor-rr-acceptor character of the ligands. With respect to the embedded heme group of the 02-carrying hemoproteins, monoimidazoleheme appears in the most favorable situation to bind carbon monoxide. Comparison with T h e hydrophobic environment of the heme group in hemoproteins has led us and others (see, for instance, Caughey et al., 1965;Kassner, 1972Kassner, , 1973 to think that a nonaqueous solution may be a more suitable medium for iron-porphyrin studies. We have previously discussed the preparation of bare, i.e. free of axial ligands, deuteroheme in benzene, and its coordination by weak or strong field ligands (Brault and Rougee, 1974a-c) and by carbon monoxide (Rougee and Brault, 1973). W e present here a more complete study of the coordinating properties of deuteroheme in the presence of both a weak or strong field ligand (L) and carbon monoxide. The purpose of this study is to determine to what extent the fixation of a given ligand (or C O ) alters the reactivity of heme toward CO (or a ligand). Our results give information on the unusual behavior of CO among other ligands and the very high affinity of five-coordinate hemoproteins such as myoglobin and hemoglobin toward CO. We conclude that hemoproteins which bind CO with the maximum affinity hitherto reported (3-5 X IO8 M -' ) are as relaxed, according to the concept of Perutz et al. ( I974a,b), as free, unconstrained monoimidazoleheme in solution. Experimental SectionSolvents and chemicals were of the purest available grade. Chlorodeuterohemin dimethyl ester was synthesized according to routine procedure. The reduced form will be hereafter called heme.Argon, grade U, was purchased from Air-Liquide, and pure carbon monoxide (CO) was from Matheson. Diluted CO in nitrogen, grade U (respectively, 1.08 X 0.98 X lo-', and 1.01 X in volume), was specially supplied by Air-Liquide.Ultraviolet (uv), visible, and near-infrared (ir) (up to 3000 nm) spectra were recorded using a Beckman DKU

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The interaction of deuteroporphyrin with dimyristoylphosphatidylcholine unilamellar vesicles of various sizes (ranging from 38 to 222 nm) has been studied using a stopped flow with fluorescence detection. Beside the kinetics of porphyrin incorporation into vesicles, the transfer of porphyrin from vesicles to human serum albumin has been investigated both experimentally and theoretically. The effects of both vesicle and albumin concentrations indicate that the transfer proceeds through the aqueous phase. It is governed by the rate of incorporation of porphyrin into the outer vesicle hemileaflet (kon), by the exit to the bulk aqueous medium (koff), and by the association (kas) and dissociation (kdis) constants relative to albumin. In both systems studied, a slower transbilayer flip-flop accounts for the biphasic character of the kinetics. This model is strongly supported by the effects of vesicle size, temperature, and cholesterol. The dependence of kon on the vesicle size indicates that the incorporation is diffusion controlled. The constant koff is found to be closely coupled to the phase state of the bilayer. The transbilayer flip-flop rate constant is approximately the same in both directions (approximately 0.4 s-1 at 32 degrees C and pH 7.4). It is strongly affected by the presence of cholesterol in vesicles and by the temperature, with a sharp enhancement around the phase transition. With the exception of very small vesicles obtained by sonication, no influence of the vesicle size on the flip-flop rate was observed. An accelerating effect of tetrahydrofuran, used to improve the solubility of porphyrin, has been noted. Steady-state measurements and kinetics results were in excellent agreement. The interest of systems involving albumin as a scavenger to extract important rate constants, is emphasized.