Relative Propensities of Cytochrome c Oxidase and Cobalt Corrins for

Nov 8, 2017 - Department of Environmental and Occupational Health, Graduate School of Public Health, The University of Pittsburgh, Pittsburgh, Pennsyl...
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Relative Propensities of Cytochrome c Oxidase and Cobalt Corrins for Reaction with Cyanide and Oxygen: Implications for Amelioration of Cyanide Toxicity Quan Yuan, Linda L. Pearce,* and Jim Peterson* Department of Environmental and Occupational Health, Graduate School of Public Health, The University of Pittsburgh, Pittsburgh, Pennsylvania 15219, United States S Supporting Information *

ABSTRACT: In aqueous media at neutral pH, the binding of two cyanide molecules per cobinamide can be described by two formation constants, Kf1 = 1.1 (±0.6) × 105 M−1 and Kf2 = 8.5 (±0.1) × 104 M−1, or an overall cyanide binding constant of ∼1 × 1010 M−2. In comparison, the cyanide binding constants for cobalamin and a fully oxidized form of cytochrome c oxidase, each binding a single cyanide anion, were found to be 7.9 (±0.5) × 104 M−1 and 1.6 (±0.2) × 107 M−1, respectively. An examination of the cyanide-binding properties of cobinamide at neutral pH by stopped-flow spectrophotometry revealed two kinetic phases, rapid and slow, with apparent second-order rate constants of 3.2 (±0.5) × 103 M−1 s−1 and 45 (±1) M−1 s−1, respectively. Under the same conditions, cobalamin exhibited a single slow cyanidebinding kinetic phase with a second-order rate constant of 35 (±1) M−1 s−1. All three of these processes are significantly slower than the rate at which cyanide is bound by complex IV during enzyme turnover (>106 M−1 s−1). Overall, it can be understood from these findings why cobinamide is a measurably better cyanide scavenger than cobalamin, but it is unclear how either cobalt corrin can be antidotal toward cyanide intoxication as neither compound, by itself, appears able to out-compete cytochrome c oxidase for available cyanide. Furthermore, it has also been possible to unequivocally show in head-to-head comparison assays that the enzyme does indeed have greater affinity for cyanide than both cobalamin and cobinamide. A plausible resolution of the paradox that both cobalamin and cobinamide clearly are antidotal toward cyanide intoxication, involving the endogenous auxiliary agent nitric oxide, is suggested. Additionally, the catalytic consumption of oxygen by the cobalt corrins is demonstrated and, in the case of cobinamide, the involvement of cytochrome c when present. Particularly in the case of cobinamide, these oxygen-dependent reactions could potentially lead to erroneous assessment of the ability of the cyanide scavenger to restore the activity of cyanideinhibited cytochrome c oxidase.

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INTRODUCTION While only recently approved for use in the U.S., (hydroxo)cobalamin (Cb) has been accepted to be a safe and effective cyanide antidote for some years in Europe, with the central cobalt(III) ion detoxifying cyanide by directly binding the anion.1 As supplied, the Cyanokit contains Cb in solid form that must first be dissolved (5 g of solid in ∼200 mL of saline) before it can be intravenously infused (∼15 mL per min for almost 15 min in adults), rendering Cb a less than ideal antidote for such a quick-acting toxicant as cyanide. The biological precursor, cobinamide (Cbi), contains the same macrocyclic corrin ring as Cb, but it lacks the 5,6-dimethylbenzimidazole ribonucleotide tail (Figure 1). Cbi has been reported to have a measurably greater affinity for cyanide than Cb in aqueous solution,2 resulting in efforts to develop Cbi and similar corrinoids as cyanide-detection systems;3 in vivo, Cbi is reported to be 3−11 times more efficacious than Cb as a cyanide antidote.4,5 Clearly, it is advantageous that each Cbi molecule may bind two cyanide anions compared with only one by Cb, but the relevant binding constants and kinetics of formation have not been adequately determined and reported © 2017 American Chemical Society

under physiologically relevant conditions. It remains generally accepted that the principle acute toxic effect of cyanide is to inhibit cytochrome c oxidase (complex IV of the mitochondrial electron-transport chain), especially in tissues of the central nervous system.6,7 Therefore, a comparison of the relative affinity of cyanide for cytochrome c oxidase compared to that of Cbi and Cb should be informative. The binding of cyanide anion to the heme a3-CuB binuclear active site of complex IV is not simple.8 In particular, binding of cyanide to the fully reduced or fully oxidized enzyme cannot account for the observed inhibition, and it has long been realized9,10 that one or more transient, partially reduced species, formed during enzymatic turnover, may be responsible for the most rapid cyanide binding. It has further been proposed that for the facile binding of cyanide to heme a3, both non-ligandbinding electron-transfer centers (CuA and heme a) must first be reduced,11 while the optimal status of CuB remains uncertain. In this investigation, we have compared the binding Received: October 3, 2017 Published: November 8, 2017 2197

DOI: 10.1021/acs.chemrestox.7b00275 Chem. Res. Toxicol. 2017, 30, 2197−2208

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Chemical Research in Toxicology

antidote binding sites in a manner governed by the prevailing equilibrium constants is untenable.

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EXPERIMENTAL SECTION

Reagents. All reagents were ACS grade, or better, used without further purification, and, unless stated to the contrary, purchased from Aldrich or Sigma. Argon gas (99.998%) was obtained from Matheson, and sodium dithionite, 87% minimum assay (+H2O), was from EM Science. Cobinamide was prepared by acid hydrolysis of hydroxocobalamin, purified essentially by reported chromatographic procedures; the purity of the final product was assessed by comparison of its electronic absorption characteristics with those of published spectra.14−16 Cobinamide concentrations were determined as the bis(aquo) Cbi2+ (i.e., Co(III) form at acidic pH; see the caption of Figure 1 for an explanation of structural abbreviations) using the extinction coefficient 28 mM−1 cm−1 at 348 nm.17 Enzyme. Cytochrome c oxidase was prepared as previously described18 from intact bovine heart mitochondria using a modified Harzell−Beinert procedure (without the preparation of Keilin− Hartree particles). The enzyme was determined to be spectroscopically pure if the 444/424 nm ratio for the reduced enzyme was 2.2 or higher. Derivatives were prepared in 50 mM potassium phosphate, 1 mM sodium EDTA, and 0.1% lauryl maltoside, pH 7.4−7.8, to concentrations of 5−80 μM (in enzyme). Enzyme concentrations were determined as total heme a using the differential (absorption) extinction coefficient of Δε604 = 12 mM−1 cm−1 for the reduced minus oxidized spectra of the mammalian enzyme. Concentrations throughout are given on a per enzyme concentration basis (i.e., [heme a]/2). Assays. Ferrocytochrome c:O2 oxidoreductase activity was spectrophotometrically determined by employing the high ionic strength method of Sinjorgo et al.19 Using this assay, we routinely obtain a turnover number with respect to cytochrome c of 340 (±30) s−1 (260 μM O2, 0.1 M sodium phosphate, 0.1% lauryl maltoside, pH 7.4, 22 °C), similar to that of the bovine enzyme isolated from a variety of tissues by others.19 Oxygen consumption kinetics were measured polarographically using a catalytic amount of cytochrome c (6 μM) and 5 mM sodium ascorbate as the reductant. Reactions were carried out at room temperature in 0.1 M potassium phosphate buffer, 0.1% lauryl maltoside, pH 7.4, 22 °C, at an initial oxygen concentration of ∼230 μM. All kinetic time courses for oxygen consumption (and ferrocytochrome c oxidation) were essentially linear in the range 10− 60 s. Where required, rates were estimated from the linear-region slopes of the oxygen (or ferrocytochrome c) concentration versus time plots without applying corrections. Titrations. For determinations of cyanide binding equilibria, all solutions were maintained in “capped” (septum sealed, with head volumes minimized) vessels and transfers were made with gastight syringes. Titration procedures were repeated multiple times with varying combinations of equipment and personnel to ensure that the results were not biased by systematic errors arising from technique. Relatively concentrated cyanide solutions (in 0.167 M borate, pH 11) were titrated into much larger volumes of complex IV, Cbi, or Cb solutions (in 50 mM phosphate, pH 7.4) to maintain neutrality in reaction mixtures. Deviation from pH 7.4 was verified to be less than 0.05 pH units upon completion of all titrations. In the case of complex IV and Cb, binding constants were estimated by plotting the log of the saturation data (Y/(1 − Y)) determined from the absorbance changes, where Y represents the proportion of sites occupied by cyanide, versus the log of the free cyanide (i.e., HCN) concentrations. Linear fits to this data gave plots with slopes of 1 (indicating no cooperativity), and the y intercepts yielded the binding constants for cyanide to heme a3 or Cb. Analysis of the more complicated Cbi data is described in the Results. Instrumentation. Electronic absorption spectra were measured and photometric determinations were made using Shimadzu UV1650PC and UV-2501PC spectrophotometers. Rates of electron transfer from reduced cytochrome c to cytochrome c oxidase under saturating [O2] (260 μM at 20 °C) were followed at 550 nm. A Clark-

Figure 1. Structures of cobinamide (Cbi) and cobalamin (Cb). Common features are the corrin ring carrying a single negative charge and the central cobalt ion, usually isolated as Co(III), although Co(II) is also common, and Co(I) is less so. The axial ligands X and Y are exchangeable, but in the case of Cb around neutral pH, Y is almost always the N3 of its 5,6-benzimidazolyl moiety. Note also that the phosphate group of Cb provides an additional negative charge compared to Cbi derivatives.33 (Nomenclature: In this article, involving only aqueous solutions, we do not explicitly indicate the presence of aquo ligands and write the total charge on the complex due to the axial ligands, cobalt ion, corrin ring, and phosphate group; e.g., Cbi2+ ⇒ bis(aquo)Co(III); Cbi(OH)+ ⇒ aquohydroxoCo(III); Cbi(CN)2 ⇒ bis(cyano)Co(III); Cb+ ⇒ aquoCo(III); CbCN ⇒ cyanoCo(III); CbOH ⇒ hydroxoCo(III), etc.)

affinities of cyanide to Cbi and Cb with that of complex IV, at physiological hydrogen ion concentrations, under both static and the more pertinent enzymatic turnover conditions. It has been known for some considerable time that many Co(II) complexes have a tendency to react with oxygen.12,13 In keeping with this general observation, we have discovered that Cb and Cbi catalyze oxygen reduction by electron donors available in vivo. This reaction certainly has important implications with regard to the possible toxicity of cobalt corrins (and other similar compounds), but of more immediate concern to us was that it interfered in experiments where we were attempting to directly measure the reversal of cyanide inhibition of complex IV by Cbi using either routine polarographic or spectrophotometric methods for the enzyme inhibition kinetics. We have been able to overcome this difficulty by performing a nonstandard set of kinetic experiments, with appropriate controls, from which the effects of Cb and Cbi on complex IV activity and its inhibition by cyanide could be calculated. Overall, the findings provide confirmation of Cbi being a better cyanide antidote than Cb, but the underlying mechanisms accounting for the antidotal activity of these and other cobalt-containing cyanide-scavenging compounds appear to be more complicated than has typically been supposed. In particular, any notion that cyanide simply becomes redistributed between complex IV and any available 2198

DOI: 10.1021/acs.chemrestox.7b00275 Chem. Res. Toxicol. 2017, 30, 2197−2208

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Chemical Research in Toxicology

Figure 2. Consumption of oxygen by Cb and Cbi. Reactions were carried out in 100 mM potassium phosphate buffer, pH 7.4, 1 mM EDTA, and 0.1% lauryl maltoside at 25 °C. Transfers were made using gastight syringes, and the samples were contained in septum-sealed vessels. (A) Ferrocytochrome c (35 μM) oxidation by complex IV (0.75 nM) monitored by following absorbance decreases at 550 nm (solid trace). Addition of Cbi(OH)+ (to 50 μM) to the turnover mixture results in an apparent increase in cytochrome c oxidation (dashed trace). (B) Oxygen consumption rates catalyzed by Cbi (0−25 μM, closed circles) in the presence of ferrocytochrome c (6 μM) or Cb (10−20 μM, closed squares) and excess sodium ascorbate (10 mM) followed by a Clark electrode. (C) Effect of varying the ratio of cytochrome c (1.5−12 μM) to Cbi (6 μM) on the oxygen consumption rate using a Clark electrode. Addition of sodium cyanide (to 20 μM) to the 1:1 cytochrome c:Cbi sample resulted in a 33% decrease in the oxygen consumption rate. type electrode (Rank Brothers), calibrated using saturated sodium bisulfate (0% calibration) and air-saturated buffer (100% calibration), was employed to carry out the oxygen uptake experiments. The oxygen-depletion experiments performed under a closed-system configuration of the Clark-type electrode showed linearity from 100 to ∼12% oxygen levels over a 3 min period. X-band EPR spectra were recorded and analyzed using the SpinCount program (graciously provided by Prof. M. Hendrich, Department of Chemistry, Carnegie Mellon University) as previously described.20 Rapid-mixing kinetics were measured with an Applied Photophysics stopped-flow/laser-flash spectrometer (LKS.60-SX.18MV-R system), and the data were fit using PC Pro-K software (!SX.18MV) provided by the manufacturer.

presence of excess ascorbate adding only a catalytic amount of cytochrome c. Unfortunately, however, addition of Cbi(OH)+ also led to increased oxygen consumption during the kinetic experiments with complex IV (data not shown). The rate of oxygen loss in the presence of excess ascorbate (10 mM) and cytochrome c (6 μM) while varying the amount of Cbi(OH)+ added, but in the complete absence of any complex IV, showed a linear dependence on the concentration of Cbi(OH)+ (Figure 2B). The process is clearly catalytic, as oxygen consumption continued to depletion even when the Cbi(OH)+ concentration was much lower than the initial oxygen concentration. Importantly, we did not observe any similarly rapid changes in oxygen concentration with Cbi(OH)+ and ascorbate alone; the addition of cytochrome c clearly accelerated oxygen consumption, with maximal reaction rates achieved when the ratio of cytochrome c to Cbi(OH)+ was near 1:1 (Figure 2C). Subsequent assay with Amplex Red demonstrated the formation of hydrogen peroxide in the ascorbate/Cbi(OH)+/ cytochrome c solutions, but less than 15% of the consumed oxygen was detected as hydrogen peroxide, suggesting additional processes, such as O2 → H2O2 → 2H2O. A more limited set of experiments was performed with Cb rather than Cbi (Figure 2B) resulting in two interesting observations. First, the rate of oxygen consumption in the presence of Cb was 13fold slower than the comparable reaction with Cbi. Second, the absence of cytochrome c from the reaction mixture did not affect the rate of oxygen consumption by Cb (data not shown). Comparative Binding of Cyanide to Fully Oxidized Complex IV versus Cobinamide at Physiological pH. The equilibrium constants for the binding of cyanide to Cbi(OH)+ have previously been reported under acidic conditions (pH ∼ 2)2 and also under basic conditions (pH 8−12),21 leading to estimates of the overall constant for the reaction Cbi(OH)+ + 2CN− ⇆ Cbi(CN)2 from ∼1019 to 1022 M−2 by independent groups.2,21,22 This reaction and consequent formulation of the equilibrium constant originated with George et al.22 and was then adopted by Hayward et al.2 based upon assumptions that were reasonable at the time. In a seminal study more than a decade later, however, Reenstra and Jencks23 determined that in the case of the reaction with Cb, HCN rather than CN− must be the attacking species around neutrality. Subsequently,

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RESULTS Cobalamin (Cb) and Cobinamide (Cbi) Consume Oxygen. It has been previously reported11 that the highest cyanide affinity for cytochrome c oxidase (complex IV) occurs when the enzyme is turning over, that is, in the presence of excess ferrocytochrome c and oxygen. Consequently, while it would obviously seem desirable to evaluate the ability of cyanide scavenging agents to reactivate cyanide-inhibited complex IV under turnover conditions, this is not a straightforward proposition. Addition of complex IV (to 2.0 nM) to a solution of ferrocytochrome c (35 μM) in the presence of oxygen (0.25 mM) at 25 °C leads to loss of absorbance at 550 nm, indicative of cytochrome c oxidation (Figure 2A, solid trace); this is a standard assay procedure for complex IV activity. Surprisingly, the rate of cytochrome c oxidation increased dramatically with the addition of Cbi(OH)+ (to 50 μM) (Figure 2A, broken trace). Noting that no cyanide was present in these experiments, the results demonstrate oxidation of ferrocytochrome c by one or more Cbi-derived species. Electronic absorption measurements (not shown) indicated that ferrocytochrome c could reduce Cbi(OH)+ to Cbi+ (i.e., Co(III) → Co(II) with the loss of a hydroxyl ion), and subsequent EPR measurements confirmed this finding (see below). Because this was the case, we concluded that monitoring the disappearance of ferrocytochrome c as a measure of electron transfer activity by complex IV in the presence of added Cbi(OH)+ would lead to erroneous results. In an effort to circumvent this issue, we subsequently followed the disappearance of oxygen using a Clark electrode in the 2199

DOI: 10.1021/acs.chemrestox.7b00275 Chem. Res. Toxicol. 2017, 30, 2197−2208

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Chemical Research in Toxicology Baldwin et al.24 confirmed this to also be the case for the Cbi reaction. Furthermore, in aqueous solution at pH 7.4, Cbi exists principally as Cbi(OH)+ (i.e., the aquohydroxoCo(III) complex)24 and the mono(cyano) adduct exists as Cbi(CN)+ (i.e., the aquocyanoCo(III) complex).25 Consequently, since the pKa of hydrocyanic acid is 9.3, the two stepwise equilibrations in question are better described by eqs 1 and 2 Cbi(OH)+ + HCN ⇆ Cbi(CN)+ + H 2O; K f1 = [Cbi(CN)+ ]/([Cbi(OH)+ ][HCN])

(1)

Cbi(CN)+ + HCN ⇆ Cbi(CN)2 + H3O+ ; K f2 = [Cbi(CN)2 ][H+]/([Cbi(CN)+ ][HCN])

(2)

As we are primarily interested in comparing the binding affinity of different systems at pH 7.4, we may further simplify this by ignoring the hydrogen ion concentration and defining an effective formation constant K′f2 = [Cbi(CN)2]/([Cbi(CN)+][HCN + CN−]) ≈ [Cbi(CN)2]/([Cbi(CN)+][HCN]), which gives K′f = [Cbi(CN)2]/([Cbi(OH)+][HCN]2) for the overall reaction under neutral conditions. Also, from a mechanistic perspective, since the substitution of an aquo ligand by cyanide is more likely than displacement of a substitution-inert hydroxo ligand,24 the first reaction (eq 1) is almost certainly a multistep process. The likely sequence is as follows: (i) HCN acting as a nitrogen donor (isocyanide) displaces the aquo ligand of Cbi(OH)+, followed by (ii) deprotonation of the bound HCN and (iii) rearrangement to the cyanohydroxoCo(III) intermediate where the coordinate bond is now through the CN− carbon.23 At some point during this sequence, (iv) the trans hydroxo ligand must acquire a proton from water to generate the required aquocyanoCo(III) product. Of course, the second reaction (eq 2) requires only steps (i) through (iii) for completion. The constants Kf1 and K′f2 for the binding of cyanide to Cbi(OH)+ were determined at pH 7.4 in 50 mM phosphate buffer by slowly titrating aliquots of NaCN solution into a capped cuvette and following the resulting changes in the visible-region absorption spectrum. The starting spectrum exhibited a maximum at 494 nm (Figure 3, main panel, dotdashed trace) arising from the Cbi(OH)+ species. During the earliest part of the titration, an increase in absorbance was observed at 517 nm (Figure 3, main panel, broken trace) that we interpret to represent formation of the mono(cyano) adduct. Subsequently, as the titration progressed, a peak at 578 nm began to emerge (Figure 3, main panel, dotted and solid traces), indicating the formation of the final bis(cyano) product. As expected for overlapping spectra arising from three interconverting species, there was no well-maintained isosbestic point. However, superimposing only spectra obtained during the later stages of the titration, where about half or more of the Cbi had been converted to the bis(cyano) species, led to the observation of an approximately isosbestic point at 527 nm (Figure 3, inset). Consequently, to a very good approximation, the data in this latter restricted range can be analyzed taking only the equilibrium of eq 2 into account. The progress of titrations were monitored by following absorbance changes at 578 and 488 nm (Figure 4A, closed squares and closed circles, respectively). These particular data were obtained in the presence of 0.1% lauryl maltoside for ease of subsequent comparison with cytochrome c oxidase findings, but experiments were also performed in the absence of lauryl

Figure 3. Electronic absorption spectra of Cbi-derived mixtures of species observed during titration with cyanide at pH 7.4. Small aliquots of sodium cyanide solution in 0.167 M sodium tetraborate buffer (pH 11) were titrated into a solution of Cbi(OH)+ (40 μM in 50 mM sodium phosphate buffer, pH 7.4) using gastight syringes and a 1.00 cm path length septum-sealed cuvette at 20 °C (see Experimental Section for further details). Main panel: initial Cbi(OH)+: dot/dashed trace; 8 μM in NaCN: dashed trace; 60 μM in NaCN: dotted trace; final Cbi(CN)2: solid trace. Inset: spectra obtained at 60, 75, 90, and 200 μM NaCN.

Figure 4. Titrations of Cbi(OH)+ with cyanide at pH 7.4. (A) Small aliquots of sodium cyanide solution in 0.167 M sodium tetraborate buffer (pH 11) were titrated into a solution of Cbi(OH)+ (40 μM in 50 mM sodium phosphate buffer, pH 7.4, 1 mM in EDTA, 0.1% lauryl maltoside) using gastight syringes and a 1.00 cm path length septumsealed cuvette at 20 °C (see Experimental Section for further details). The absorbance changes were followed at two different wavelengths, 488 nm (closed circles, minimum in the difference spectrum Cbi(OH)+ − Cbi(CN)2) and 578 nm (closed squares, maximum for Cbi(CN)2). (B) Log−log plots of absorbance at 578 nm versus cyanide concentration comparing the present data (closed squares) with that reported by Marques et al.21 (open squares); the slopes of the linear fits and their intercepts on the y-axis (off scale) are the same within experimental uncertainty.

maltoside (data not shown). Working at 488 nm (rather than at 494 nm, the maximum for the Cbi(OH)+ species) minimized overlap with the spectrum of the final Cbi(CN)2 product. Even if they are scaled (not shown), the data sets at the two wavelengths were never quite mirror images of each other, reflecting the greater interference (i.e., spectral overlap) of the mono(cyano) intermediate’s spectrum with that of the starting Cbi(OH)+ species at 488 nm than with the Cbi(CN)2 product at 578 nm. Consequently, starting with 40 μM Cbi(OH)+ and using only the data obtained at 578 nm in the absorbance range 0.15−0.45, where only eq 2 needs to be considered, 2200

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relevant set of our own (Figure 4B). The offset between the two data sets is due to variability in the selection of absorbance baseline positions in the two studies and is not significant. The important comparisons are between the slopes of the linear fits and their extrapolated values on the ordinate axis, with the latter being positive and well outside the plot limits shown. In neither case do these key parameters differ appreciably between the two studies, meaning that any derived equilibrium constants should essentially agree, certainly to within about an order of magnitude. In this study, we have restricted ourselves to consideration of the reaction at pH 7.4 under which conditions HCN is the reactive cyanide species. The earlier authors were interested in a much wider pH range and chose to write their expressions in terms of cyanide anion concentration rather than HCN. To compare the present determinations of the equilibrium constant with the earlier literature values, we must recalculate our results in terms of [CN−] (determined from the known pKa = 9.3 at 20 °C) and also divide by the hydrogen ion concentration at pH 7.4. This treatment yields 1020 to 1021 for the net equilibrium constant for Cbi(CN)2 formation, in agreement with the previous findings. In another more recent study, a value of 1.8 × 106 M−1 was reported27 for the equilibrium constant governing the reaction between Cbi(CN)+ and Cbi(CN)2 at pH 7.5. These authors also appear to have expressed the stability constant in terms of calculated [CN−]; therefore, to compare with our findings expressed in terms of [HCN + CN−] (≈[HCN] at neutral pH), we must divide by ∼102, which gives 1.8 × 104 M−1, in reasonable agreement with our estimate for K′f2 of 3.6 × 104 M−1. As the binding of cyanide to cytochrome c oxidase (complex IV) can be highly preparation dependent,8 the association constant for the binding of cyanide to the oxidized form of complex IV as currently isolated was determined in 50 mM potassium phosphate buffer (0.05% lauryl maltoside) and a pH of 7.4 at 20 °C by titrating the enzyme with sodium cyanide and following the resulting electronic absorption changes. Cyanide solutions were freshly made for each titration experiment in 0.167 M borate buffer, pH 10.5, for addition in small aliquots (relative to the total volume of phosphatebuffered solution). The binding of cyanide to oxidized (i.e., as isolated) complex IV is somewhat complicated because there are multiple fast and slow cyanide binding forms of the enzyme in any given preparation. To overcome this problem, we allowed the solutions to reach equilibrium by waiting approximately 10 min after cyanide additions before collecting any data points and confirmed stability of the signals by taking repeated absorbance readings. In addition, during the titration experiments we also observed a small amount of the enzyme being converted to the reduced form (Soret maximum at 444 nm). The addition of cyanide to the oxidized form of complex IV shifts the Soret from ∼422 to 428 nm, resulting in a difference spectrum with a minimum at 411 nm and a maximum at 433 nm (not shown). To avoid any interference from the small amount of reduced complex IV produced during the titration of the oxidized enzyme, we chose to use the absorbance changes at 436 nm (the isosbestic wavelength of oxidized and reduced heme a28) to follow the cyanide binding to heme a3. We calculated the formation constant, Kf_IV = [heme a3-CN]/([heme a3][HCN]), for cyanide binding to the current preparation of fully oxidized complex IV from log−log plots (see Experimental Section) to be 1.6 (±0.2) × 107 M−1. On the basis solely of consideration of the relative equilibium affinity constants that we have determined for cyanide binding

concentrations of Cbi(CN)2 at different points in the titration (X) were determined as X = (AX − Ainitial)/(Afinal − Ainitial) × 40 μM. Concentrations of Cbi(CN)+ (Y) were then estimated as Y = 40 − XμM, enabling free [HCN] to be calculated as [NaCN added] − 2X − Y. These parameters were used in conjunction with eq 2 to find the effective formation constant K′f2 at pH 7.4 and 20 °C to be 8.5 × 104 M−1 in the presence of lauryl maltoside and 3.6 × 104 M−1 in the absence of the detergent. Next, we applied the hyperbolic sine procedure described by Asuero26 for the determination of two related equilibrium constants of similar magnitude from spectrophotometric titration data. Since K′f2 was already known, it was possible to essentially fix this value and fit the data to determine Kf1 alone, which was estimated to be 1.1 × 105 M−1 with lauryl maltoside and 2.9 × 104 M−1 without lauryl maltoside. Therefore, at pH 7.4 and 20 °C, the overall equilibrium constant for the formation of Cbi(CN)2 is given by Kf1·K′f2 ∼ 9.6 × 109 M−2 (with lauryl maltoside) and Kf1·K′f2 ∼ 1.0 × 109 M−2 (without lauryl maltoside). For comparison, we also measured the formation constant of Cb(CN) (i.e., the cyanoCo(III) adduct of cobalamin) at pH 7.4 and 20 °C in the presence of lauryl maltoside, obtaining Kf_Cb = 7.8(±0.5) × 104 M−1. It is noteworthy that this present result differs considerably from the Cb(CN) formation constant previously reported to be ∼1012 by Hayward et al.2 The earlier authors were working at pH 4.8, and they were following the recombination of the photodissociated complex in a reaction exhibiting a half time of several hours; whereas in our titrations, the ligand substitutions were complete within 1−2 min of the cyanide solution additions. Unsurprisingly, these reactions under acidic (