Crystal structure of an RNA aptamer bound to thrombin

Aptamers, an emerging class of therapeutics, are DNA or RNA molecules that are selected to bind molecular targets that range from small organic compounds to large proteins. All of the determined structures of aptamers in complex with small molecule targets show that aptamers cage such ligands. In structures of aptamers in complex with proteins that naturally bind nucleic acid, the aptamers occupy the nucleic acid binding site and often mimic the natural interactions. Here we present a crystal structure of an RNA aptamer bound to human thrombin, a protein that does not naturally bind nucleic acid, at 1.9 A resolution. The aptamer, which adheres to thrombin at the binding site for heparin, presents an extended molecular surface that is complementary to the protein. Protein recognition involves the stacking of single-stranded adenine bases at the core of the tertiary fold with arginine side chains. These results exemplify how RNA aptamers can fold into intricate conformations that allow them to interact closely with extended surfaces on non-RNA binding proteins.     

Crystal structural analysis of human serum albumin complexed with hemin and fatty acid

Background

Human serum albumin (HSA) is an abundant plasma protein that binds a wide variety of hydrophobic ligands including fatty acids, bilirubin, thyroxine and hemin. Although HSA-heme complexes do not bind oxygen reversibly, it may be possible to develop modified HSA proteins or heme groups that will confer this ability on the complex.

Results

We present here the crystal structure of a ternary HSA-hemin-myristate complex, formed at a 1:1:4 molar ratio, that contains a single hemin group bound to subdomain IB and myristate bound at six sites. The complex displays a conformation that is intermediate between defatted HSA and HSA-fatty acid complexes; this is likely to be due to low myristate occupancy in the fatty acid binding sites that drive the conformational change. The hemin group is bound within a narrow D-shaped hydrophobic cavity which usually accommodates fatty acid; the hemin propionate groups are coordinated by a triad of basic residues at the pocket entrance. The iron atom in the centre of the hemin is coordinated by Tyr161.

Conclusion

The structure of the HSA-hemin-myristate complex (PDB ID 1o9x) reveals the key polar and hydrophobic interactions that determine the hemin-binding specificity of HSA. The details of the hemin-binding environment of HSA provide a structural foundation for efforts to modify the protein and/or the heme molecule in order to engineer complexes that have favourable oxygen-binding properties.

     

Two alternative conformations of S-adenosyl-L-homocysteine bound to Escherichia coli DNA adenine methyltransferase and the implication of conformational changes in regulating the catalytic cycle

The crystal structure of the Escherichia coli DNA adenine methyltransferase (EcoDam) in a binary complex with the cofactor product S-adenosyl-L-homocysteine (AdoHcy) unexpectedly showed the bound AdoHcy in two alternative conformations, extended or folded. The extended conformation represents the catalytically competent conformation, identical to that of EcoDam-DNA-AdoHcy ternary complex. The folded conformation prevents catalysis, because the homocysteine moiety occupies the target Ade binding pocket. The largest difference between the binary and ternary structures is in the conformation of the N-terminal hexapeptide ((9)KWAGGK(14)). Cofactor binding leads to a strong change in the fluorescence of Trp(10), whose indole ring approaches the cofactor by 3.3A(.) Stopped-flow kinetics and AdoMet cross-linking studies indicate that the cofactor prefers binding to the enzyme after preincubation with DNA. In the presence of DNA, AdoMet binding is approximately 2-fold stronger than AdoHcy binding. In the binary complex the side chain of Lys(14) is disordered, whereas Lys(14) stabilizes the active site in the ternary complex. Fluorescence stopped-flow experiments indicate that Lys(14) is important for EcoDam binding of the extrahelical target base into the active site pocket. This suggests that the hexapeptide couples specific DNA binding (Lys(9)), AdoMet binding (Trp(10)), and insertion of the flipped target base into the active site pocket (Lys(14)).     

Electron transfer complexes of cytochrome c peroxidase from Paracoccus denitrificans containing more than one cytochrome

According to the model proposed in previous papers [Pettigrew, G. W., Prazeres, S., Costa, C., Palma, N., Krippahl, L., and Moura, J. J. (1999) The structure of an electron-transfer complex containing a cytochrome c and a peroxidase, J. Biol. Chem. 274, 11383-11389; Pettigrew, G. W., Goodhew, C. F., Cooper, A., Nutley, M., Jumel, K., and Harding, S. E. (2003) Electron transfer complexes of cytochrome c peroxidase from Paracoccus denitrificans, Biochemistry 42, 2046-2055], cytochrome c peroxidase of Paracoccus denitrificans can accommodate horse cytochrome c and Paracoccus cytochrome c(550) at different sites on its molecular surface. Here we use (1)H NMR spectroscopy, analytical ultracentrifugation, molecular docking simulation, and microcalorimetry to investigate whether these small cytochromes can be accommodated simultaneously in the formation of a ternary complex. The pattern of perturbation of heme methyl and methionine methyl resonances in binary and ternary solutions shows that a ternary complex can be formed, and this is confirmed by the increase in the sedimentation coefficient upon addition of horse cytochrome c to a solution in which cytochrome c(550) fully occupies its binding site on cytochrome c peroxidase. Docking experiments in which favored binary solutions of cytochrome c(550) bound to cytochrome c peroxidase act as targets for horse cytochrome c and the reciprocal experiments in which favored binary solutions of horse cytochrome c bound to cytochrome c peroxidase act as targets for cytochrome c(550) show that the enzyme can accommodate both cytochromes at the same time on adjacent sites. Microcalorimetric titrations are difficult to interpret but are consistent with a weakened binding of horse cytochrome c to a binary complex of cytochrome c peroxidase and cytochrome c(550) and binding of cytochrome c(550) to the cytochrome c peroxidase that is affected little by the presence of horse cytochrome c in the other site. The presence of a substantial capture surface for small cytochromes on the cytochrome c peroxidase has implications for rate enhancement mechanisms which ensure that the two electrons required for re-reduction of the enzyme after reaction with hydrogen peroxide are delivered efficiently.     

Structure of complex III with bound cytochrome c in reduced state and definition of a minimal core interface for electron transfer

In cellular respiration, cytochrome c transfers electrons from cytochrome bc(1) complex (complex III) to cytochrome c oxidase by transiently binding to the membrane proteins. Here, we report the structure of isoform-1 cytochrome c bound to cytochrome bc(1) complex at 1.9 A resolution in reduced state. The dimer structure is asymmetric. Monovalent cytochrome c binding is correlated with conformational changes of the Rieske head domain and subunit QCR6p and with a higher number of interfacial water molecules bound to cytochrome c(1). Pronounced hydration and a "mobility mismatch" at the interface with disordered charged residues on the cytochrome c side are favorable for transient binding. Within the hydrophobic interface, a minimal core was identified by comparison with the novel structure of the complex with bound isoform-2 cytochrome c. Four core interactions encircle the heme cofactors surrounded by variable interactions. The core interface may be a feature to gain specificity for formation of the reactive complex.     

Detection and comparison of specific hemin binding by Porphyromonas gingivalis and Prevotella intermedia.

A radioligand assay was designed to detect and compare specific hemin binding by the periodontal anaerobic black-pigmenting bacteria (BPB) Porphyromonas gingivalis and Prevotella intermedia. The assay included physiological concentrations of the hemin-binding protein rabbit serum albumin (RSA) to prevent self-aggregation and nonspecific interaction of hemin with cellular components. Under these conditions, heme-starved P. intermedia cells (two strains) expressed a single binding site species (4,100 to 4,600 sites/cell) with a dissociation constant (Kd) of 1.0 x 10(-9) M. Heme-starved P. gingivalis cells (two strains) expressed two binding site species; the higher-affinity site (1,000 to 1,500 sites/cell) displayed a Kd of between 3.6 x 10(-11) and 9.6 x 10(-11) M, whereas the estimated Kd of the lower-affinity site (1.9 x 10(5) to 6.3 x 10(5) sites/cell) ranged between 2.6 x 10(-7) and 6.5 x 10(-8) M. Specific binding was greatly diminished in heme-replete cells of either BPB species and was not displayed by iron-replete Escherichia coli cells, which bound as much hemin in the absence of RSA as did P. intermedia. Hemin binding by BPB was reduced following treatment with protein-modifying agents (heat, pronase, and N-bromosuccinimide) and was blocked by protoporphyrin IX and hemoglobin but not by Congo red. Hemopexin also inhibited bacterial hemin binding. These findings indicate that both P. gingivalis and P. intermedia express heme-repressible proteinaceous hemin-binding sites with affinities intermediate between those of serum albumin and hemopexin. P. gingivalis exhibited a 10-fold-greater specific binding affinity and greater heme storage capacity than did P. intermedia, suggesting that the former would be ecologically advantaged with respect to heme acquisition.     

On the binding of tRNA to Escherichia coli RNA polymerase. Interactions between the core enzyme, DNA and tRNA

We have investigated the interplay between the binding of tRNA and DNA to core RNA polymerase. We show that the monomer core enzyme can bind stably to either DNA or tRNA, whereas the dimer core can fix both DNA and tRNA in a stable ternary complex. We have examined the kinetics of the exchange between DNA and tRNA bound to the core enzyme. DNA bound to monomer core can be rapidly displaced by tRNA without prior dissociation of the core from the DNA. Similarly tRNA bound to the core can be displaced by DNA without prior dissociation of the tRNA. We confirm the result of Hinkle and Chamberlin [J. Mol. Biol. 70, 157-185 (1972)] that, in contrast, the core enzyme must first dissociate from one DNA molecule before it can transfer to another DNA. As this dissociation is very slow we suggest that, in vivo, the tRNA can act as a 'porter' providing the core enzyme with a more kinetically favourable path to transfer from one DNA site to another.     

Characterisation of binding of the methoxyacrylate inhibitors to mitochondrial cytochrome c reductase

The three E-beta-methoxyacrylate (MOA) inhibitors oudemansin A, strobilurin A and MOA stilbene [3-methoxy-2(2-styrylphenyl)propenic acid-methylester], which differ by more than one order of magnitude in their binding affinity to the mitochondrial ubihydroquinone:cytochrome c oxidoreductase (bc1 complex), bind to a site that is not identical to the binding site for ubihydroquinone, the substrate of the outer ubiquinone reaction site (Qo centre). Although the ubihydroquinone molecule is still bound in the presence of the MOA inhibitors, its electrons cannot be transferred to the iron-sulfur centre. A shift of the relative position of the ubihydroquinone molecule in the reaction centre due to a conformational distortion of cytochrome b induced by the binding of the MOA inhibitor seems to be the reason for the blocked electron transfer. Further analysis shows that ubihydroquinone affects the Kd values of all three MOA inhibitors tested: the values are raised by a constant factor of two, although the inhibitors bind with quite different affinity. The iron-sulfur protein is not involved in the binding of the MOA inhibitors. These results have direct implications for the proper use of MOA inhibitors in experiments designed to analyse the structure/mechanism relationship in cytochrome c reductase. In particular, point mutations recently described in MOA-inhibitor-resistant mutants can no longer be taken to affect necessarily the ubihydroquinone binding site.     

Ligand binding to ferrocytochrome c at high pH.

Ferrocytochrome c has been shown to bind two molecules of CO at pH 14. The second CO is thought to be bound only when the cytochrome c molecule is denatured, and once bound appears to be spectrally silent. Insolubilization of native cytochrome c prevents the binding of the second CO molecule. A scheme is proposed to explain these observations based on evidence from static titrations and flash-photolysis experiments, use of carboxymethyl cytochrome c and insoluble cytochrome c, and use of cyanide instead of CO as a ligand.     

Monitoring complex formation in the blood-coagulation cascade using aptamer-coated SAW sensors

Specific binding of the anticoagulants heparin and antithrombin III to the blood clotting cascade factor human thrombin was recorded as a function of time with a Love-wave biosensor array consisting of five sensor elements. Two of the sensor elements were used as references. Three sensor elements were coated with RNA or DNA aptamers for specific binding of human thrombin. The affinity between the aptamers and thrombin, measured using the biosensor, was within the same range as the value of K(D) measured by filter binding experiments. Consecutive binding of the thrombin inhibitors heparin, antithrombin III or the heparin-antithrombin III complex to the immobilized thrombin molecules, and binding of a ternary complex of heparin, anithrombin III, and thrombin to aptamers was evaluated. The experiments showed attenuation of binding to thrombin due to heparin-antithrombin III complex formation. Binding of heparin activated the formation of the inhibitory complex of antithrombin III with thrombin about 2.7-fold. Binding of the DNA aptamer to exosite II appeared to inhibit heparin binding to exosite I.     

Detection system based on the conformational change in an aptamer and its application to simple bound/free separation

Aptamers are good molecular recognition elements for biosensors. Especially, their conformational change, which is induced by the binding to the target molecule, enables the development of several types of useful detection systems. We applied this property to bound/free separation, which is a crucial process for highly sensitive detection. We designed aptamers which change their conformation upon binding to the target molecule and thereby expose a single-strand bearing the complementary sequence to the capture probe immobilized onto the support. We named the designed aptamers "capturable aptamers" and the capture probe "capture DNA". Three capturable aptamers were designed based on the PrP aptamer, which binds to prion protein. One of these capturable aptamers was demonstrated to recognize prion protein and change its conformation upon binding to it. A detection system using this designed capturable aptamer for prion protein was developed. Capturable aptamers and capture DNA allow us to perform simple bound/free separation with only one target ligand.     

Binding of arylazidocytochrome c derivatives to beef heart cytochrome c oxidase: cross-linking in the high- and low-affinity binding sites

Two arylazidocytochrome c derivatives, one modified at lysine-13 and the second modified at lysine-22, were reacted with beef heart cytochrome c oxidase. The lysine-13 modified arylazidocytochrome c was found to cross-link both to the enzyme and with lipid bound to the cytochrome c oxidase complex. The lysine-22 derivative reacted only with lipids. Cross-linking to protein was through subunit II of the cytochrome c oxidase complex, as first reported by Bisson et al. [Bisson, R., Azzi, A., Gutweniger, H., Colonna, R., Monteccuco, C., & Zanotti, A. (1978) J. Biol. Chem. 253, 1874]. Binding studies show that the cytochrome c derivative covalently bound to subunit II was in the high-affinity binding site for the substrate. Evidence is also presented to suggest that cytochrome c bound to the lipid was in the low-affinity binding site [as defined by Ferguson-Miller et al. [Ferguson-Miller, S., Brautigan, D. L., & Margoliash, E. (1976) J. Biol. Chem. 251, 1104]]. Covalent binding of the cytochrome c derivative into the high-affinity binding site was found to inhibit electron transfer even when native cytochrome c was added as a substrate. Inhibition was almost complete when 1 mol of the Lys-13 modified arylazidocytochrome c was covalently bound to the enzyme per cytochrome c oxidase dimer (i.e., congruent to 280 000 daltons). Covalent binding of either derivative with lipid (low-affinity site) had very little effect on the overall electron transfer activity of cytochrome c oxidase. These results are discussed in terms of current theories of cytochrome c-cytochrome c oxidase interactions.     

Formation of a complex between yeast L-lactate dehydrogenase (cytochrome b2) and cytochrome c. Ultracentrifugal and gel chromatographic analyses.

Yeast L-lactate dehydrogenase formed a stable complex with cytochrome c in weakly alkaline solution of low ionic strength. The binding ratio of cytochrome c to the enzyme depended on whether free cytochrome c was present: In the presence of a micromolar concentration of cytochrome c the enzyme formed a complex with about two molecules of cytochrome c, whereas the enzyme was in a 1:1 molecular complex after removal of free cytochrome c. This suggests that the binding of one molecule of cytochrome c changes the affinity of the other binding site on the enzyme for cytochrome c. The enzyme consists of four presumably identical subunits, each containing a binding site for cytochrome c. Thus, present data confirm the concept of negative cooperativity between the subunits of the enzyme molecule in their interaction with cytochrome c.     

Anion and ionic strength effects upon the oxidation of cytochrome c by cytochrome c oxidase

Citrate and other polyanion binding to ferricytochrome c partially blocks reduction by ascorbate, but at constant ionic strength the citrate-cytochrome c complex remains reducible; reduction by TMPD is unaffected. At a constant high ionic strength citrate inhibits the cytochrome c oxidase reaction competitively with respect to cytochrome c, indicating that ferrocytochrome c also binds citrate, and that the citrate-ferrocytochrome c complex is rejected by the binding site at high ionic strength. At lower ionic strengths, citrate and other polyanions change the kinetic pattern of ferrocytochrome c oxidation from first-order towards zero-order, indicating preferential binding of the ferric species, followed by its exclusion from the binding site. The turnover at low cytochrome c concentrations is diminished by citrate but not the Km (apparent non-competitive inhibition) or the rate of cytochrome a reduction by bound cytochrome c. Small effects of anions are seen in direct measurements of binding to the primary site on the enzyme, and larger effects upon secondary site binding. It is concluded that anion-cytochrome c complexes may be catalytically competent but that the redox potentials and/or intramolecular behaviour of such complexes may be affected when enzyme-bound. Increasing ionic strength diminishes cytochrome c binding not only by decreasing the 'association' rate but also by increasing the 'dissociation' rate for bound cytochrome c converting the 'primary' (T) site at high salt concentrations into a site similar kinetically to the 'secondary' (L) site at low ionic strength. A finite Km of 170 microM at very high ionic strength indicates a ratio of K infinity m/K 0 M of about 5000. It is proposed that anions either modify the E10 of cytochrome C bound at the primary (T) site of that they perturb an equilibrium between two forms of bound c in favour of a less active form.     

Interaction of cytochrome c with tRNA and other polynucleotides

Interaction between tRNA and other polynucleotides with cytochrome c was studied by visible spectroscopy, fluorescence spectroscopy and gel mobility shift assay in view of the recently reported important regulatory role of tRNA in cytochrome c mediated apoptotic pathway. Visible spectroscopy showed perturbation in the heme binding environment in cytochrome c with tRNA binding. Fluorescence titrations indicated that cytochrome c binds to different polynucleotides with differing affinities. A weak binding was observed with single stranded polyribonucleotides and polydeoxyribonuleotides and strong binding with tRNA and double stranded DNA as indicated by extent of fluorescence quenching and binding constants. Calculation of thermodynamic binding parameters from fluorescence titrations indicated that three molecules of cytochrome c bound with one tRNA molecule with binding constant of 1.9?×?10(6)?M(-1). The perturbation of cytochrome c structure caused by the binding of tRNA could be affecting its role in mediating apoptosis.     

Characterization of the interaction of cytochrome c and mitochondrial ubiquinol-cytochrome c reductase

Characterization of the steady state kinetics of reduction of horse ferricytochrome c by purified beef ubiquinol-cytochrome c reductase, employing 2,3-dimethoxy-5-methyl-6-decylbenzoquinol as reductant, has shown that: 1) the dependence of the reaction on quinol and on ferricytochrome c concentration is consistent with a ping-pong mechanism; 2) the pH optimum of the reaction is near 8.0; 3) the effect of ionic strength on the apparent Km and the TNmax of the reaction for the native cytochrome c is small, and at higher cytochrome c concentrations substrate inhibition is observed; 4) the effect of ionic strength on the kinetic parameters for the reaction of 4-carboxy-2,6-dinitrophenyllysine 27 horse cytochrome c is much larger than for the native protein; and 5) competitive product inhibition is also observed with a Ki consistent with the binding affinity of ferrocytochrome c for Complex III, as determined by gel filtration. In addition, direct binding measurements demonstrated that ferricytochrome c binds more tightly than the reduced protein to Complex III under low ionic strength conditions and that under these conditions more than one molecule of cytochrome c is bound per molecule of Complex III. Exchange of Complex III into a nonionic detergent decreases this excess nonspecific binding. Measurement of the rates of dissociation of the oxidized and reduced 1:1 complexes of cytochrome c and Complex III by stopped flow was consistent with the disparity of binding affinities, the dissociation rate constant for ferrocytochrome c being about 5-fold higher than that for the ferric protein. A model which accounts for the properties of this system is described, assuming that cytochrome c bound to noncatalytic sites on the respiratory complex decreases the catalytic site binding constant for the substrate.     

G-quadruplex-forming aptamer enhances the peroxidase activity of myoglobin against luminol

Aptamers can control the biological functions of enzymes, thereby facilitating the development of novel biosensors. While aptamers that inhibit catalytic reactions of enzymes were found and used as signal transducers to sense target molecules in biosensors, no aptamers that amplify enzymatic activity have been identified. In this study, we report G-quadruplex (G4)-forming DNA aptamers that upregulate the peroxidase activity in myoglobin specifically for luminol. Using in vitro selection, one G4-forming aptamer that enhanced chemiluminescence from luminol by myoglobin''s peroxidase activity was discovered. Through our strategy—in silico maturation, which is a genetic algorithm-aided sequence manipulation method, the enhancing activity of the aptamer was improved by introducing mutations to the aptamer sequences. The best aptamer conserved the parallel G4 property with over 300-times higher luminol chemiluminescence from peroxidase activity more than myoglobin alone at an optimal pH of 5.0. Furthermore, using hemin and hemin-binding aptamers, we demonstrated that the binding property of the G4 aptamers to heme in myoglobin might be necessary to exert the enhancing effect. Structure determination for one of the aptamers revealed a parallel-type G4 structure with propeller-like loops, which might be useful for a rational design of aptasensors utilizing the G4 aptamer-myoglobin pair.     

Hemin Regulation of Hemoglobin Binding by Porphyromonas gingivalis

Hemoglobin binding to chemostat-grown hemin-excess and hemin-limited cells of Porphyromonas gingivalis W50, and to cells of the avirulent, beige-pigmenting variant W50/BE1, was quantified. Hemin-excess W50 bound more hemoglobin than hemin-limited W50, mirroring the hemin-binding ability of these cells [Microb Ecol Health Dis 7:9–15, 1994]. In contrast to hemin, hemoglobin binding was not enhanced by sodium dithionite. The hemoglobin-binding capacity of hemin-excess W50/BE1 was below that of hemin-limited W50 and only observed under oxidizing conditions. Scatchard analysis revealed similar affinity constants for hemin-excess and hemin-limited W50, and confirmed a lower binding maximum for the latter. Hemin-excess W50/BE1 displayed cooperative binding of hemoglobin. These differences in binding were reflected in the binding of a horse radish peroxidase-conjugated hemoglobin (HHRPO) in a dot-blot assay. However, neither the 32-kDa hemin-binding protein, nor its 19-kDa heat-modified form, from either hemin-limited W50 or hemin-excess W50/BE1, bound this conjugate. These data indicate that hemoglobin binding by P. gingivalis is hemin-regulated and occurs via a mechanism different from hemin binding. Received: 2 June 1997 / Accepted: 4 August 1997     

Spectroscopic analysis of the interaction between cytochrome c and cytochrome c oxidase

Complex formation between cytochrome c oxidase and cytochrome c perturbs the optical absorption spectrum of heme c and heme a in the region of the alpha-, beta, and gamma-bands. The perturbations have been used to titrate cytochrome c oxidase with cytochrome c. A stoichiometry of one molecule of cytochrome c bound per molecule of cytochrome c oxidase is obtained (1 heme c per heme aa3). In contrast, a stoichiometry of 2:1 was found earlier using a gel-filtration method (Rieder, R., and Bosshard, H.R. (1978) J. Biol. Chem. 253, 6045-6053). From the result of the spectrophotometric titration and from the wavelength position of the perturbation signals it is concluded that cytochrome c oxidase contains only a single binding site for cytochrome c which is close enough to heme a to function as an electron transfer site. The second site detected earlier by the gel-filtration method must be remote from this electron transfer site. Scatchard plots of the titration data are curvilinear, possibly indicating interactions between cytochrome c-binding sites on adjacent monomers of dimeric cytochrome c oxidase. The relationship between cytochrome c binding and the reaction of cytochrome c oxidase with ferrocytochrome c is discussed.     

Properties of cytochrome c modified by attachment to a carbohydrate polymer.

By enzymic digestion of the polysaccharide part of the covalent complex between cytochrome c and Sephadex G-200, a new water-soluble cytochrome c derivative is obtained (called cytochrome cr). Measurement of the free amino groups of this derivative indicates that on average the molar ratio between cytochrome c and polysaccharide is close to 1. Chemical determination of the sugar content gives a value of approx. 24000 for the molecular weight of cytochrome cr. On these bases the soluble cytochrome cr complex may be thought of as a folded protein to which a long polysaccharide tail is covalently bound. The functional behaviour of cytochrome cr is much more similar to that of the native molecule than to that of the insoluble complex (cytochrome ci). In particular the kinetics of the reaction of cytochrome cr and cytochrome cn (native) with ascorbate, ferrocyanide-ferricyanide, O2 and cytochrome c oxidase were investigated in considerable detail. The results of these experiments, together with the observation that the insoluble complex of cytochrome c is a very poor substrate of cytochrome c oxidase [Colosimo, Brunori & Antonini (1976) Biochem. J. 153, (657-661], indicate that hindrance effects constraining the approach between cytochrome cr and its oxidase are of greater importance than specific chemical modifications in determining the functional behavior of the protein.