Heme P460 of hydroxylamine oxidoreductase of Nitrosomonas. Reaction with CO and H2O2

Hydroxylamine oxidoreductase (HAO) of Nitrosomonas catalyzes the dehydrogenation of NH2OH and subsequent addition of oxygen to form nitrite. HAO contains c hemes and the CO-binding heme P460 in a 7:1 ratio; dehydrogenation of NH2OH involves passage of electrons to P460 and then c hemes. We now report that electrons rapidly pass from c hemes of HAO to the P460 center and then to H2O2. This conclusion is supported by (a) inhibition of c heme oxidation with CO and (b) loss of H2O2-oxidizability of ferrous c hemes following specific destruction of heme P460. Reaction of ferrous P460 with H2O2 is rate-limiting. Activation of dioxygen for N-oxidation by ferrous HAO may involve the two-electron reduction of O2 by P460. The reaction of ferrous HAO with H2O2 was studied as it may reveal aspects of the mechanism of activation of dioxygen. Reaction of ferrous heme P460 with CO is slow and with low affinity as compared with other hemoproteins. Values for reaction of CO with enzyme were: k1, 1.1 X 10(-3) M-1 s-1 and Kd, 12 microM.     

High-pressure stopped-flow spectrometry at low temperatures

A stopped-flow instrument operating over temperature and pressure ranges of +30 to -20 degrees C and 10(-3) to 2 kbar , respectively, is described. The system has been designed so that it can be easily interfaced with many commercially available spectrophotometers of fast response time, with the aid of quartz fiber optics. The materials used for the construction are inert, metal free and the apparatus has proven to be leak free at temperatures as low as -20 degrees C under a pressure of 2 kbar . The performance of the instrument was tested by measuring the rate of reduction of cytochrome c with sodium dithionite and the 2,6-dichloroindophenol/ascorbate reaction. The dead time of the system has been evaluated to be 20, 50, and congruent to 100 ms in water at 20 degrees C, in 40% ethylene glycol/water, and at 20 degrees C and -15 degrees C, respectively. These values are rather pressure independent up to 2 kbar . Application of the bomb was demonstrated using the cytochrome c peroxidase/ethyl peroxide reaction. This process occurred in two phases and an increase in pressure decreased the rates of reactions indicating two positive volumes of activation (delta V not equal to app (fast) = 9.2 +/- 1.5 ml X mol-1; delta V not equal to app (slow) = 14 +/- 1.5 ml X mol-1, temperature 2 degrees C). The data suggest that the fast reaction could involve a hydrophobic bond, whereas the slow process could be associated with a stereochemical change of the protein. The problem of temperature equilibrium for high-pressure experiments is also discussed.     

Activation thermodynamics of the binding of carbon monoxide to horseradish peroxidase. Role of pressure, temperature and solvent

The kinetics at 423 nm of the binding of carbon monoxide to ferrous horseradish peroxidase were studied as a function of three parameters: pressure (1-1200 bar), temperature (34 to -20 degrees C) and solvent (water, 40% ethylene glycol, 50% methanol) using a high-pressure stopped-flow apparatus. By using transition state theory the thermodynamic quantities delta V, delta S and delta H were determined under these different experimental conditions and were found to be greatly modulated by the physico-chemical parameters of the media. The results suggest that the macroscopic thermodynamic response is mainly controlled by the solvent. By adjusting two variables (among T, P, solvent), it is possible either to amplify or to cancel out the effect of the third.     

Contribution of water to free energy of hydrolysis of pyrophosphate

The energy of hydrolysis of phosphate compounds varies depending on whether they are in solution or bound to the catalytic site of enzymes. With the purpose of simulating the conditions at the catalytic site, the observed equilibrium constant for pyrophosphate hydrolysis (Kobsd) was measured in aqueous mixtures of dimethyl sulfoxide, ethylene glycol, or polymers of ethylene glycol. The reaction was catalyzed by yeast inorganic pyrophosphatase at 30 degrees C. All the cosolvents used promoted a decrease of Kobsd. Polymers of ethylene glycol were more effective than dimethyl sulfoxide or ethylene glycol in decreasing Kobsd. The higher the molecular weight of the polymer, the lower the value of Kobsd. A decrease in Kobsd from 346 M (delta G degree obsd = -3.5 kcal mol-1) to 0.1 M (delta G degree obsd = 1.3 kcal mol-1) was observed after the addition of 50% (w/v) poly(ethylene glycol) 8000 to a solution containing 0.9 mM MgCl2 and 1 mM Pi at pH 8.0. The association constants of Pi and pyrophosphate for H+ and Mg2+ were measured in presence of different ethylene glycol concentrations in order to calculate the Keq for hydrolysis of different ionic species of pyrophosphate. A decrease in all the Keq was observed. The results are interpreted according to the concept that the energy of hydrolysis of phosphate compounds depends on the different solvation energies of reactants and products.     

The influence of temperature and osmolyte on the catalytic cycle of cytochrome c oxidase.

The influence of temperature on cytochrome c oxidase (CCO) catalytic activity was studied in the temperature range 240-308 K. Temperatures below 273 K required the inclusion of the osmolyte ethylene glycol. For steady-state activity between 278 and 308 K the activation energy was 12 kcal x mol-1; the molecular activity or turnover number was 12 s-1 at 280 K in the absence of ethylene glycol. CCO activity was studied between 240 and 277 K in the presence of ethylene glycol. The activation energy was 30 kcal x mol-1; the molecular activity was 1 s-1 at 280 K. Ethylene glycol inhibits CCO by lowering the activity of water. The rate limitation in electron transfer (ET) was not associated with ET into the CCO as cytochrome a was predominantly reduced in the aerobic steady state. The activity of CCO in flash-induced oxidation experiments was studied in the low temperature range in the presence of ethylene glycol. Flash photolysis of the reduced CO complex in the presence of oxygen resulted in three discernable processes. At 273 K the rate constants were 1500 s-1, 150 s-1 and 30 s-1 and these dropped to 220 s-1, 27 s-1 and 3 s-1 at 240 K. The activation energies were 5 kcal.mol-1, 7 kcal.mol-1, and 8 kcal.mol-1, respectively. The fastest rate we ascribe to the oxidation of cytochrome a3, the intermediate rate to cytochrome a oxidation and the slowest rate to the re-reduction of cytochrome a followed by its oxidation. There are two comparisons that are important: (a). with vs. without ethylene glycol and (b). steady state vs. flash-induced oxidation. When one makes these two comparisons it is clear that the CCO only senses the presence of osmolyte during the reductive portion of the catalytic cycle. In the present work that would mean after a flash-induced oxidation and the start of the next reduction/oxidation cycle.     

Redox potentials in hydro-organic media at normal and subzero temperatures. Ferro-ferricyanide and cytochrome c as models.

Redox potentials of ferro-ferricyanide and cytochrome c were measured in water/ethylene glycol and water/dimethylsulfoxide (volume ratio from 100/0 to 50/50) between 25 and -25 degrees C. For both systems, the midpoint potential decreases in the presence of organic solvents and increases by cooling. The magnitude of these variations is larger in dimethylsulfoxide than in ethylene glycol; moreover in the same solvent mixture it is larger with ferro-ferricyanide than with cytochrome c, so that the difference between the redox potentials of these two systems can be strongly affected and even reversed. While in pure water (cacodylate buffer pH 7.0, NaCl 0.1 M) they are respectively +388 and +265 mV, in 50% dimethylsulfoxide at 25 degrees C they decrease to +112 and +208 mV. Reduction of cytochrome c by ferro-ferricyanide, in this mixture, is then expected and was indeed observed. On the other hand, as (deltaE/deltaT)T, (E being the redox potential) is higher for ferro-ferricyanide than for cytochrome c, the oxidative power of the former for the latter is expected to increase as temperature decreases. This effect was observed in 50% ethylene glycol at -16 degrees C. Organic solvents and large temperature variations appear then as powerful perturbants of redox reactions. Their effects should be taken into account in studies of redox reactions carried out in cooled hydro-organic media.     

Liposome formation of egg lecithin and its interaction with iodine

The sonicated dispersion of egg lecithin (phosphatidylcholine) in water forms 1:1 molecular complex with iodine, when its concentration is above 1.6 X 10(-5) M. The thermodynamic and spectrophotometric properties of this complex have been determined. The thermodynamic values are: K (25 degrees C) = 1.6 X 10(3) 1 X mol-1, delta G degrees = -18.4 KJ X mol-1, delta H degrees = -27.4 KJ X mol-1 and delta S degrees = -30.0 J X mol-1 X deg-1. The complex shows two absorption bands: one at 293 nm, which is the charge transfer band and the other at 370 nm, which is the blue shifted visible iodine band at 460 nm in water.     

Activation parameters for the carbonic anhydrase II-catalyzed hydration of CO2

We have determined the activation parameters of kcat and kcat/Km for the carbonic anhydrase II-catalyzed hydration of CO2. The enthalpy and entropy of activation for kcat is 7860 +/- 120 cal mol-1 and -3.99 +/- 0.42 cal mol-1 K-1, respectively, for the human enzyme. Results for the bovine enzyme were statistically indistinguishable from those of the human enzyme. The entropy of activation of kcat for the human enzyme was further decomposed into partially compensating electrostatic(es) (delta S*es = +15.1 cal mol-1 K-1) and nonelectrostatic(nes) (delta S*nes = -19.1 cal mol-1 K-1) terms. Computer simulations of a formal kinetic mechanism for carbonic anhydrase II-catalyzed CO2 hydration show that 82% of the temperature effect on kcat can be attributed to the temperature effect on the intramolecular proton transfer step. The reported activation parameters are consistent with a substantial enzyme or active site solvent conformational change in the transition state of the intramolecular proton transfer step, and is consistent with the mechanism of proton transfer proposed by Venkatasubban and Silverman (Venkatasubban, K. S., and Silverman, D. N. (1980) Biochemistry 19, 4984-4989).     

Transient kinetics of the interaction of 1,N6-ethenoadenosine 5'-triphosphate with myosin subfragment 1 under normal and cryoenzymic conditions: a comparison with adenosine 5'-triphosphate

The kinetics of the interaction of the fluorescent analogue 1,N6-ethenoadenosine 5'-triphosphate (epsilon-ATP) with myosin subfragment 1 (S1) were studied at 15 and -7.5 degrees C with 40% ethylene glycol as cryosolvent. Two techniques were used: fluorescence stopped flow and rapid flow-quench. When S1 is mixed with epsilon-ATP in a stopped-flow apparatus, biphasic fluorescence transients are obtained which are difficult to assign. Chemical sampling by the rapid-flow-quench method led to the chemical identity and the kinetics of interconversion of key intermediates, and by this method the optical signals were assigned and information about the cleavage and release of products was obtained. The data were interpreted by a shortened form of the Bagshaw-Trentham scheme for myosin adenosinetriphosphatase: M + ATP K1 in equilibrium M.ATP k2----M*.ATP k3 in equilibrium k3 M**.ADP.Pi k4----M + ADP + Pi The constants obtained were compared with those for ATP under identical conditions. In agreement with Rosenfeld and Taylor [Rosenfeld, S. S., & Taylor, E. W. (1984) J. Biol. Chem. 259, 11920-11929] we find that epsilon-ATP is bound tightly to S1 and that the chemical step is slower than with ATP. We show that the fast fluorescence transient is due to the tight binding of epsilon-ATP with K1 = 32 microM and k2 = 58 s-1 at 15 degrees C. With ATP these values are 8 microM and 16 s-1, respectively. There is a large difference in the delta H for k2: 50 kJ.mol-1 for epsilon-ATP and 119 kJ.mol-1 for ATP.(ABSTRACT TRUNCATED AT 250 WORDS)     

M?ssbauer, EPR, and optical studies of the P-460 center of hydroxylamine oxidoreductase from Nitrosomonas. A ferrous heme with an unusually large quadrupole splitting

Hydroxylamine oxidoreductase from Nitrosomonas europeae catalyzes the oxidative conversion of NH2OH to NO-2. The enzyme, Mr = 220,000, has an (alpha beta)3 subunit structure with each alpha beta subunit containing 7-8 c-type hemes and one unusual prosthetic group, termed P-460. The P-460 is also found in a Mr approximately equal to 17,000 protein (P-460 fragment). M?ssbauer spectra of the reduced P-460 groups, in hydroxylamine oxidoreductase and the fragment, exhibit nearly identical quadrupole doublets with an unusually large splitting, delta EQ = 4.21 mm/s (no ferrous heme protein is known with delta EQ greater than 2.75 mm/s). The observed isomer shift, delta = 0.96 mm/s at 4.2 K, shows that the P-460 iron is high spin ferrous. Treatment of oxidized hydroxylamine oxidoreductase with H2O2 followed by reduction or exposure of the native sample to CO led to the disappearance of both the characteristic 460 nm absorption band (epsilon = 89 mM-1 cm-1) and the delta EQ = 4.21 mm/s doublet. The iron of the oxidized P-460 fragment is high spin ferric, with M?ssbauer and EPR parameters very similar to those of metmyoglobin. Optical spectra of the reduced P-460 fragment show long wavelength bands at 650 and 688 nm which are sensitive to treatment of the fragment with reagents which react with P-460. These bands were, however, not detected in hydroxylamine oxidoreductase. The spectroscopic and chemical evidence obtained to date suggests strongly that the P-460 iron resides in a heme-like macrocycle although the presumed porphyrin must have some unusual features.     

Pressure effects on catalysis by alkaline phosphatase reconstituted in lipid bilayers

Bovine intestinal alkaline phosphatase (EC 3.1.3.1) was reconstituted into lipid bilayers by a dilution method using n-octylglucopyranoside. From the kinetic measurements at various pressures, the volume of activation (delta V not equal to) and volume change in substrate binding (delta V) were estimated for free and reconstituted ALP. The delta V not equal to and delta V values for free ALP and reconstituted ALP in the gel state liposome showed opposite tendencies (-23 ml . mol-1 [delta V not equal to], 35 ml . mol-1 [delta V] for free ALP and 27 ml . mol-1 [delta V not equal to], -36 ml . mol-1 [delta V] for reconstituted ALP, respectively), which suggest both strong desolvation effect of enzyme molecule by the surrounding lipids and drastic conformational change of the enzyme molecule by the reconstitution into liposomes.     

The cholesterol-side-chain-cleaving cytochrome P450 spin-state equilibrium. 1. Thermodynamic analysis.

We have investigated the spin-state equilibrium of adrenal mitochondrial P450scc (cholesterol-side-chain-cleaving, CYP11A1) by absorption spectroscopy in the Soret band as a function of pH and temperature. The van't Hoff plot of the high-spin/low-spin equilibrium is not linear and is shifted towards high spin by lowering the pH. This non-linearity resolves clearly into two phases when the temperature range is extended from 37 degrees C to -20 degrees C using ethylene glycol as anti-freeze cosolvent. This enabled us to measure the enthalpy and entropy changes which are delta HA = 0.7 kJ.mol-1 and delta SA = 5J.K-1.mol-1 at low temperatures and delta HB = -42 kJ.mol-1 and delta SB = -152 J.K-1.mol-1 at high temperatures. The transition temperature, Tbreak, between both phases decreases as a function of pH. The experimental data can be fitted by a minimal reactional model comprising a temperature dependent conformational transition and two ionisation steps (one for each conformation), the pK of which is 1.5 +/- 0.5 higher in the low-temperature conformation. The deduced conformational equilibrium is affected by physiological effectors: Tbreak depends on the nature of the substrate intermediate and on the presence of the physiological electron donor, adrenodoxin.     

Acylation of subtilisin A by aryl esters: contribution to rate limitation by a physical step preceding general acid-base catalysis

The specificity ratios kc/Km = k for subtilisin A catalyzed hydrolysis of five aryl esters of N-(methoxycarbonyl)-L-Phe (McPhe) were determined at pH 7.03 and its pD equivalent. The ratios are independent of the electronic properties of the leaving group substituent. Kinetic solvent isotope effects, Dk, increase from about 0.9 to 1.3 as leaving group ability decreases from p-nitrophenolate to p-methoxyphenolate. The k of N-(methoxycarbonyl)-L-phenylalanine p-nitrophenyl ester (NPE) with native enzyme exhibits a strong temperature dependence; delta H* = 87 +/- 3 kJ mol-1 and delta S* = 148 +/- 14 J K-1 mol-1 at 25 degrees C (H2O). The Dk with this substrate is 1.36 at 13.6 degrees C, declines to 0.89 at 25 degrees C, and then increases to 1.04 at 39.4 degrees C. Above neutral pH(D), with McPhe NPE as substrate, the dependence of k is for the dissociated form of a single base of pKapp = 7.38 +/- 0.03 in H2O and 7.67 +/- 0.03 in D2O. The pKapp values are apparently those of the uncomplexed native protein. By contrast, k of 3-phenylpropanoic acid (Prop) p-nitrophenyl ester exhibits a weaker temperature dependence; delta H* = 20 kJ mol-1 and delta S* = -90 J K-1 mol-1 (H2O) at 25 degrees C. The Dk are larger than those for McPhe NPE, decreasing from 1.99 at 20.5 degrees C to 1.74 at 46.1 degrees C. These results, combined with those of previous studies, are consistent with limitation of k by at least two processes.(ABSTRACT TRUNCATED AT 250 WORDS)     

Dynamics of carbon monoxide recombination to fully reduced cytochrome c oxidase in plant mitochondria after low-temperature flash photolysis.

Rebinding of CO to reduced cytochrome c oxidase in plant mitochondria has been monitored optically at 590-630 nm after flash photolysis at low temperature from 160 to 200 K. (1) Under 100%-CO saturation, CO rebinding exhibits a four-step mechanism. The thermodynamic parameters of the first phase have been determined; its activation energy, Ea1, is 38.9 kJ.mol-1 and its enthalpy, delta H+/-1, and entropy, delta S+/-1, of activation are respectively 37.5 kJ.mol-1 and -75.8J.mol-1.K-1. (2) When the CO concentration is decreased to 0.2%, rebinding still occurs according to a four-step mechanism. The rate constant of the first phase is CO-concentration-independent. Under non-saturating conditions there is only one CO molecule per occupied site. The rebinding mechanism does not require additional CO molecules to be present in the haem pocket. (3) Dual-wavelength scanning experiments failed to detect optical forms correlated with the resolved phases. (4) Results are discussed with respect to previous work related to CO rebinding to mammalian cytochrome c oxidase and myoglobin.     

Equilibrium and kinetic studies on the binding of gluconolactone to almond beta-glucosidase in the absence and presence of glucose

The binding of glucono-1,5-lactone (gluconolactone) with almond beta-glucosidase was studied at pH 5.0 and 25 degrees C, in the absence and presence of glucose, by monitoring the enzyme fluorescence as a probe. From the results of fluorometric titration, the dissociation constant Kd and the maximum fluorescence intensity increase (percent) of the enzyme-gluconolactone complex relative to the enzyme alone, delta Fmax, were determined to be 12.7 microM and 14.7%, respectively. From the study of the temperature dependence of Kd, delta G degrees, delta H degrees and delta S degrees for the binding were evaluated to be -6.7 kcal mol-1, -3.5 kcal mol-1, and 10.8 e.u. (cal mol-1 deg-1), respectively, at 25 degrees C. The analysis of the fluorometric titration data in the presence of glucose revealed that these ligands bind competitively to the enzyme, probably at the same site. The results of a stopped-flow kinetic study are consistent with the following two-step mechanism: (formula; see text) which indicates that gluconolactone (L) and the enzyme (E) transiently form a loosely bound complex, ELtr (k-1/k+1 = 4.5 mM), in the first rapid bimolecular association step, and ELtr is converted into a more tightly bound complex EL (k+2 = 94 s-1, k-2 = 0.36 s-1) in the subsequent slow unimolecular process. The fluorescence intensity increase occurs solely in the latter step.     

Pressure effects on substrate activation phenomena in the alpha-chymotrypsin-catalyzed hydrolysis of p-nitrophenyl acetate

With and without p-chlorophenol as an activator, the rates of hydrolysis of p-nitrophenyl acetate catalyzed by alpha-chymotrypsin were measured at pressures up to 2 kbar at 25 degrees C. From the pressure dependence of the rate constant (kcat)A and (kcat)0 of the product formation with and without an activator, the activation volumes (delta V not equal to cat)A and (delta not equal to cat)0 were +2 and -6 +/- 1 cm3.mol-1. From the pressure dependence of the equilibrium constant (KA) of incorporation of p-chlorophenol into the enzyme, the volume change (delta VA) was -10 +/- 1 cm3.mol-1. The mechanisms of the substrate activation are discussed in terms of the activation and reaction volumes.     

Effects of pH, ionic strength, and temperature on activation by calmodulin an catalytic activity of myosin light chain kinase

The reversible association of Ca42+-calmodulin with the inactive catalytic subunit of myosin light chain kinase results in the formation of the catalytically active holoenzyme complex [Blumenthal, D. K., & Stull, J. T. (1980) Biochemistry 19, 5608--5614]. The present study was undertaken in order to determine the effects of pH, temperature, and ionic strength on the processes of activation and catalysis. The catalytic activity of myosin light chain kinase, when fully activated by calmodulin, exhibited a broad pH optimum (greater than 90% of maximal activity from pH 6.5 to pH 9.0), showed only a slight inhibition by moderate ionic strengths (less than 20% inhibition at mu = 0.22), and displayed a marked temperature dependence (Q10 congruent to 2; Ea = 10.4 kcal mol-1). Thermodynamic parameters calculated from Arrhenius plots indicate that the Gibb's energy barrier associated with the rate-limiting step of catalysis is primarily enthalpic. The process of kinase activation by calmodulin had a narrower pH optimum (pH 6.0--7.5) than did catalytic activity, was markedly inhibited by increasing ionic strength (greater than 70% inhibition at mu = 0.22), and exhibited nonlinear van't Hoff plots. Between 10 and 20 degrees C, activation was primarily entropically driven (delta S degrees congruent to 40 cal mol-1 deg-1; delta H degrees = -900 cal mol-1), but between 20 and 30 degrees C, enthalpic factors predominated in driving the activation process (delta S degrees congruent to 10 cal mol-1 deg-1; delta H degrees = -9980 cal mol-1). The apparent change in heat capacity (delta Cp) accompanying activation was estimated to be -910 cal mol-1 deg-1. On the basis of these data we propose that although hydrophobic interactions between calmodulin and the kinase are necessary for the activation of the enzyme, other types of interactions such as hydrogen bonding, ionic, and van der Waals interactions also make significant and probably obligatory contributions to the activation process.     

The site and mechanism of dioxygen reduction in bovine heart cytochrome c oxidase

The site and mechanism of dioxygen reduction in cytochrome c oxidase from bovine heart muscle have been investigated. The rate of cytochrome c2+ oxidation by O2 is shown to be affected by several factors: 1) pH, with optima at 5.65 and 6.0, 2) temperature between 0 and 29 degrees C, with E alpha = 13 kcal mol-1, 3) D2O exchange, with a reduction in rate of 50% or more at the pH optima, and 4) the addition of ethylene glycol or glycerol, which significantly lowers the rate. The extremely narrow (delta vCO approximately 4 cm-1) infrared stretch bands at approximately 1964 and approximately 1959 cm-1 for liganded CO are only slightly affected by factors 1-4 or by changes in the oxidation state of metals other than the heme alpha 3 iron. These results indicate a stable, unusually immobile O2 reduction site well-isolated from the external medium, a characteristic expected to be important for oxidase function. Precise stereochemical positioning of hydrogen donors adjacent to O2 liganded to heme alpha 3 iron can be expected in order to achieve the optimization of the time/distance relationships required for enzyme catalysis. These findings support a novel mechanism of O2 reduction via a hydroperoxide intermediate within a reaction pocket that experiences little change in conformation during the hydrogen and electron transfer steps.     

A jump in an Arrhenius plot can be the consequence of a phase transition. The binding of ATP to myosin subfragment 1

The temperature dependence of the kinetics of the binding of ATP to myosin subfragment-1 was studied by an ATP chase technique in a rapid-flow-quench apparatus: (formula; see text) A temperature range of 30 degrees C to -15 degrees C was obtained with ethylene glycol as antifreeze. The Arrhenius plot of k2 is discontinuous with a jump at 12 degrees C. Above the jump delta H+ = 9.5 kcal/mol, below delta H+ = 28.5 kcal/mol. Few such Arrhenius plots are recorded in the literature but they are predicted from theory. Thus, we explain our results as a phase change of the subfragment 1-ATP system at 12 degrees C. This is in agreement with certain structural studies.     

High-pressure effect on the equilibrium and kinetics of cyanide binding to chloroperoxidase

The kinetics of cyanide binding to chloroperoxidase were studied using a high-pressure stopped-flow technique at 25 degrees C and pH 4.7 in a pressure range from 1 to 1000 bar. The activation volume change for the association reaction is delta V not equal to + = -2.5 +/- 0.5 ml/mol. The total reaction volume change, determined from the pressure dependence of the equilibrium constant, is delta V degrees = -17.8 +/- 1.3 ml/mol. The effect of temperature was studied at 1 bar yielding delta H not equal to + = 29 +/- 1 kJ/mol, delta S not equal to + = -58 +/- 4 J/mol per K. Equilibrium studies give delta H degrees = -41 +/- 3 kJ/mol and delta S degrees = -59 +/- 10 J/mol per K. Possible contributions to the binding process are discussed: changes in spin state, bond formation and conformation changes in the protein. An activation volume analog of the Hammond postulate is considered.