The proton pump of heme-copper oxidases.

Proton pumping heme-copper oxidases represent the terminal, energy-transfer enzymes of respiratory chains in prokaryotes and eukaryotes. The Cu_B-heme a₃ (or heme o) binuclear center, associated with the largest subunit I of cytochrome c and quinol oxidases, is directly involved in the coupling between dioxygen reduction and proton pumping. The role of the other subunits is less clear. The following aspects will be covered in this paper:i) the efficiency of coupling in the mitochondrial aa₃ cytochrome c oxidase. In particular, the effect of respiratory rate and protonmotive force on the H⁺/e^? stoichiometry and the role of subunit IV; ii) mutational analysis of the aa₃ quinol oxidase of Bacillus subtilis addressed to the role of subunit III, subunit IV and specific residues in subunit I; iii) possible models of the protonmotive catalytic cycle at the binuclear center. The observations available suggest that H⁺/e^?coupling is based on the combination of protonmotive redox catalysis at the binuclear center and co-operative proton transfer in the protein.     

Ultrasonic studies of proton-transfer reactions at the catalytic site of alpha-chymotrypsin

Ultrasonic relaxation measurements for alpha-chymotrypsin in phosphate, sulfite and arsenate buffers exhibit a high peak of absorption at neutral pH. The analysis is based on: comparison of the relaxation measurements for the enzyme and for the zymogen and inhibited enzyme; X-ray and neutron diffraction data, and high-resolution NMR data. The ultrasonic relaxation is shown to result mainly from a proton-transfer reaction that involves the histidine at the catalytic site (His-57). The question is raised of whether the enhanced ultrasonic effect observed in the enzyme is indicative of a property that plays a part in the catalytic activity.     

Direct infrared detection of the covalently ring linked His-Tyr structure in the active site of the heme-copper oxidases

Infrared spectroscopy, isotopic labeling ([(15)N(delta,epsilon)]histidine and ring-deuterated tyrosine), synthetic model studies, and normal mode calculations are employed to search for the spectroscopic signatures of the unique, covalently linked (His N(epsilon)-C(epsilon) Tyr) biring structure in the heme-copper oxidases. The specific enzyme examined is the cytochrome bo(3) quinol oxidase of E. coli. Infrared features of histidine and tyrosine are identified in the frequency regions of imidazole and phenol ring stretching modes (1350-1650 cm(-1)) and C-H and N-H stretching modes as well as overtones and combinations (>3000 cm(-1)). Two of these, at ca. 1480 and 1550 cm(-1), and their combination tones between 3010 and 3040 cm(-1), are definitively identified with the biring structure involving H284 and Y288 in the E. coli enzyme. Studies of a synthetic analogue of the H-Y structure, 4-methylimidazole covalently linked to p-cresol, show that a feature near 1540 cm(-1) is unique to the biring structure and is absent from the infrared spectrum of 4-methylimidazole or p-cresol alone. This feature is readily detectable by infrared difference techniques, and offers a direct spectroscopic probe for potential radical production involving the H-Y structure in the O(2) reduction cycle of the oxidases.     

Mechanism and energetics of proton translocation by the respiratory heme-copper oxidases

Recent time-resolved optical and electrometric experiments have provided a sequence of events for the proton-translocating mechanism of cytochrome c oxidase. These data also set limits for the mechanistic, kinetic, and thermodynamic parameters of the proton pump, which are analysed here in some detail. The analysis yields limit values for the pK of the "pump site", its modulation during the proton-pumping process, and suggests its identity in the structure. Special emphasis is made on side-reactions that may short-circuit the pump, and the means by which these may be avoided. We will also discuss the most prominent proton pumping mechanisms proposed to date in relation to these data.     

Mechanism and energetics of proton translocation by the respiratory heme-copper oxidases

Recent time-resolved optical and electrometric experiments have provided a sequence of events for the proton-translocating mechanism of cytochrome c oxidase. These data also set limits for the mechanistic, kinetic, and thermodynamic parameters of the proton pump, which are analysed here in some detail. The analysis yields limit values for the pK of the “pump site”, its modulation during the proton-pumping process, and suggests its identity in the structure. Special emphasis is made on side-reactions that may short-circuit the pump, and the means by which these may be avoided. We will also discuss the most prominent proton pumping mechanisms proposed to date in relation to these data.     

Carboxyl group functions in the heme-copper oxidases: information from mid-IR vibrational spectroscopy

Carboxyl groups of possible functional importance in bovine and bacterial cytochrome c oxidases (CcO) are reviewed and assessed. A critical analysis is presented of available mid-infrared vibrational data that pertain to these functional carboxyl groups. These data and their interpretations are discussed in relation to current models of the mechanism of proton and electron coupling in the protonmotive CcO superfamily.     

The histidine cycle: A new model for proton translocation in the respiratory heme-copper oxidases

A model of redox-linked proton translocation is presented for the terminal heme-copper oxidases. The new model, which is distinct both in principle and in detail from previously suggested mechanisms, is introduced in a historical perspective and outlined first as a set of general principles, and then as a more detailed chemical mechanism, adapted to what is known about the chemistry of dioxygen reduction in this family of enzymes. The model postulates a direct mechanistic role in proton-pumping of the oxygenous ligand on the iron in the binuclear heme-copper site through an electrostatic nonbonding interaction between this ligand and the doubly protonated imidazolium group of a conserved histidine residue nearby. In the model this histidine residue cycles between imidazolium and imidazolate states translocating two protons per event, the imidazolate state stabilized by bonding to the copper in the site. The model also suggests a key role in proton translocation for those protons that are taken up in reduction of O₂ to water, in that their uptake to the oxygenous ligand unlatches the electrostatically stabilized imidazolium residue and promotes proton release.     

Effects of metal ions in the CuB center on the redox properties of heme in heme-copper oxidases: spectroelectrochemical studies of an engineered heme-copper center in myoglobin

The electrochemical properties of an engineered heme-copper center in myoglobin have been investigated by UV-visible spectroelectrochemistry. In the cyanide-bridged, spin-coupled heme-copper center in an engineered myoglobin, the presence of Zn(II) in the Cu(B) center raises the heme reduction potential from -85 to 49 mV vs NHE. However, in the cyanide-free, spin-decoupled derivative of the same protein, the presence of Zn(II) in the Cu(B) center exerts little influence on the heme reduction potentials (77 and 80 mV vs NHE, respectively, in the absence and in the presence of Zn(II)). Similar trends have also been observed when copper ion is present in the Cu(B) center, although on a smaller scale, due to reduction of Cu(II) to Cu(I) prior to heme reduction. These results show that the presence of a metal ion in the designed Cu(B) center has a significant effect on the redox potential of heme Fe only when the two metal centers are coupled through a bridging ligand between the two metal centers, indicating that spin coupling plays an important role in redox potential regulation. In addition, the presence of a single positively charged Cu(I) center in the Cu(B) center resulted in a much lower increase (16 mV) in heme reduction potential than that of two positively charged Zn(II) (118 mV). Therefore, the heme reduction potential must be lowered after the first electron transfer to reduce heme Fe(3+)-Cu(B)(2+) to Fe(3+)-Cu(B)(+). To raise the heme reduction potential to make the second electron transfer (i.e., reduction of Fe(3+)-Cu(B)(+) to Fe(2+)-Cu(B)(+)) to be favorable, most likely a proton or decoupling of the heme-copper center is needed in the heme-copper site. These findings provide a strong argument for a thermodynamic driving force basis for redox-regulated proton transfer in heme-copper oxidases.     

Substrate binding and the catalytic reactions in cbb3-type oxidases: The lipid membrane modulates ligand binding

Heme–copper oxidases (HCuOs) are the terminal components of the respiratory chain in the mitochondrial membrane or the cell membrane in many bacteria. These enzymes reduce oxygen to water and use the free energy from this reaction to maintain a proton-motive force across the membrane in which they are embedded. The heme–copper oxidases of the cbb₃-type are only found in bacteria, often pathogenic ones since they have a low K_m for O₂, enabling the bacteria to colonize semi-anoxic environments. Cbb₃-type (C) oxidases are highly divergent from the mitochondrial-like aa₃-type (A) oxidases, and within the heme–copper oxidase family, cbb₃ is the closest relative to the most divergent member, the bacterial nitric oxide reductase (NOR). Nitric oxide reductases reduce NO to N₂O without coupling the reaction to the generation of any electrochemical proton gradient. The significant structural differences between A- and C-type heme–copper oxidases are manifested in the lack in cbb₃ of most of the amino acids found to be important for proton pumping in the A-type, as well as in the different binding characteristics of ligands such as CO, O₂ and NO. Investigations of the reasons for these differences at a molecular level have provided insights into the mechanism of O₂ and NO reduction as well as the proton-pumping mechanism in all heme–copper oxidases. In this paper, we discuss results from these studies with the focus on the relationship between proton transfer and ligand binding and reduction. In addition, we present new data, which show that CO binding to one of the c-type hemes of CcoP is modulated by protein–lipid interactions in the membrane. These results show that the heme c-CO binding can be used as a probe of protein–membrane interactions in cbb₃ oxidases, and possible physiological consequences for this behavior are discussed.     

Tyrosine codon corresponds to topa quinone at the active site of copper amine oxidases.

The recently discovered organic cofactor of bovine serum amine oxidase, topa quinone, is an uncommon amino acid residue in the polypeptide backbone (Janes, S. M., Mu, D., Wemmer, D., Smith, A. J., Kaur, S., Maltby, D., Burlingame, A. L., and Klinman, J. P. (1990) Science 248, 981-987). The amine oxidase gene from the yeast Hansenula polymorpha has been cloned and sequenced (Bruinenberg, P. G., Evers, M., Waterham, H. R., Kuipers, J., Arnberg, A. C., and Geert, A. B. (1989) Biochim. Biophys. Acta 1008, 157-167). In order to understand the incorporation of topa quinone in eukaryotes, we have isolated yeast amine oxidase from H. polymorpha. Following protocols established with bovine serum amine oxidase, yeast amine oxidase was derivatized with [14C]phenylhydrazine, followed by thermolytic digestion and isolation of a dominant radiolabeled peptide by high pressure liquid chromatography. Comparison of resonance Raman spectra for this peptide to spectra of a model compound demonstrates that topa quinone is the cofactor. By alignment of a DNA-derived yeast amine oxidase sequence with the topa quinone-containing peptide sequence, it is found that the tyrosine codon, UAC, corresponds to topa quinone in the mature protein. In a similar manner, alignment of a tryptic peptide from bovine serum amine oxidase implicates tyrosine as the precursor to topa quinone in mammals.     

Noninvasive auto-photoreduction used as a tool for studying structural changes in heme-copper oxidases by FTIR spectroscopy

We demonstrate an efficient Fourier transform infrared (FTIR) spectroscopic method, termed "auto-photoreduction," that uses anaerobic photo-induced internal electron transfer to monitor reaction-initiated changes of heme-copper oxidases. It can be applied without the use of either expensive electrochemical equipment, or caged compounds, which cause significant background signals. At high irradiation power, carbon monoxide is released from high-spin heme a of cytochrome c oxidase and heme o from cytochrome bo(3). Photochemistry is initiated at wavelengths <355 nm, and the photochemical action spectrum has a maximum of 290 nm for cytochrome bo(3), which is consistent with the possible intermediate involvement of tyrosinate or an activated state of tyrosine. We propose that the final electron donors are proton channel water molecules. In the pH range of 4-9, the noninvasive auto-photoreduction method yields highly reproducible FTIR redox difference spectra within a broad range, resolving a number of vibrational changes outside the amide I region (1600-1640 cm(-1)). Furthermore, it provides details of redox-induced changes in the spectral region between 1600 and 1100 cm(-1). The auto-photoreduction method should be universally applicable to heme proteins.     

Metal ion specificity at the catalytic site of yeast enolase.

A new, more gentle enzyme purification for yeast enolase was developed. A series of kinetic experiments was performed with yeast enolase where the concentration of Mg(II) is kept constant and at the Km' level; the addition of Mn(II), Zn(II), or Cu(II) gives a hyperbolic decrease in the enzyme activity. The final velocity of these mixed-metal systems is the same as the velocity obtained only with Mn(II), Zn(II), or Cu(II), respectively. The concentration of the second metal that gives half-maximal effect in the presence of Mg(II) is approximately the same as the apparent Km (Km') value measured for that cation alone. Direct binding of Mn(II) to apoenolase in the absence and presence of Mg(II) shows that Mn(II) and Mg(II) compete for the same metal site on enolase. In the presence of D-2-phosphoglycerate (PGA) and Mg(II), only a single cation site per monomer is occupied by Mn(II). Water proton relaxation rate (PRR) studies of enzyme-ligand complexes containing Mn(II) and Mn(II) in the presence of Mg(II) are consistent with Mn(II) binding at site I under both conditions. PRR titrations of ligands such as the substrate PGA or the inhibitors orthophosphate or fluoride to the enolase-Mn(II)-Mg(II) complex are similar to those obtained for the enolase-Mn(II) complex, also indicating that Mn(II) is at site I in the presence of Mg(II). High-resolution 1H and 31P NMR was used to determine the paramagnetic effect of enolase-bound Mn(II) on the relaxation rates of the nuclei of the competitive inhibitor phosphoglycolate. The distances between the bound Mn(II) and the nuclei were calculated.(ABSTRACT TRUNCATED AT 250 WORDS)     

Functional groups at the catalytic site of F1 adenosine triphosphatase

The protection of F1 ATPase by inorganic phosphate, ADP, ATP, and magnesium ion against inactivation by 1-fluoro-2,4-dinitrobenzene, 7-chloro-4-nitrobenzo-2-oxa-1,3-diazole, and 1-(ethoxycarbonyl)-2-ethoxy-1,2-dihydroquinoline, respectively, has been investigated. Dissociation equilibrium constants and rate constants for the labeling reactions have been deduced from a quantitative treatment of the kinetic data. Comparison of these dissociation constants with each other and with the corresponding literature values indicates that the essential Tyr, Arg, Lys, and Glu or Asp residues are indeed located at the catalytic site of the enzyme. Examination of the rate constants for the labeling reactions in the presence of excess inorganic phosphate, ADP, ATP, or magnesium ion, respectively, suggests that the essential phenol and amino groups are located nearer to the bound inorganic phosphate or the gamma-phosphate group than to the alpha- or beta-phosphate group of the bound ATP, that the essential guanidinium group is located nearer to the alpha- or beta-phosphate group than to the gamma-phosphate group of the bound ATP or the bound inorganic phosphate, and that the essential carboxylate group is located slightly farther away but complexed with magnesium ion which it shares with the bound inorganic phosphate. A mechanism consistent with these topographical relationships is proposed for the catalytic hydrolysis and synthesis of ATP.     

Coordination dynamics of heme-copper oxidases. The ligand shuttle and the control and coupling of electron transfer and proton translocation

Results are presented which, taken with evidence developed by others, suggest a general mechanism for the entry and binding of exogenous ligands (including O₂) at the binuclear site (Cu_B Fe _a3) of the heme-copper oxidases. The mechanism includes a ligand shuttle wherein the obligatory waystation for incoming ligands is Cu_B and the binding of exogenous ligands at this site triggers the exchange and displacement of endogenous ligands at Fe _a3. It is suggested that these ligand shuttle reactions might be functionally important in providing a coupling mechanism for electron transfer and proton translocation. Scenarios as to how this might happen are delineated.     

Role of aromatic stack pairing at the catalytic site of gelonin protein

Aromatic–aromatic interactions play an important role in the enzyme–substrate recognition mechanism and in stabilization of proteins. Gelonin – a ribosome inactivating protein (RIP) from the plant Gelonium multiflorum – belongs to type-I RIPs and shows N-glycosylation activity which has been used as a model to explain the role of aromatic–aromatic stack pairing in RIPs. RIPs have a different substrate binding site and catalytic site. Role of tyrosine residues at the binding site has already been known but the role of tyrosine residues at catalytic site is still unclear. In this study, the role of tyrosine–adenine–tyrosine aromatic stack pairing at the catalytic site was studied by in silico mutation studies using molecular dynamic simulations. Through this study we report that, despite the fact that aromatic stack pairing aids in recognition of adenine at binding site, both the tyrosine residues of stack pairing play a crucial role in the stabilization of adenine at catalytic site. In the absence of both the tyrosine residues, adenine was unstable at catalytic site that results in the inhibition of N-glycosylation activity of gelonin protein. Hence, this study highlights the importance of π–π stack pairing in the N-glycosidic activity of gelonin by determining its role in stabilizing adenine at catalytic site.     

Studies on the catalytic residues at the active site of human liver alpha-L-fucosidase

Kinetic studies and chemical modifications were performed on purified human liver alpha-L-fucosidase (alpha-L-fucoside fucohydrolase, EC 3.2.1.51) in an attempt to identify the catalytic residues at the active site. Plots of log Vmax vs. pH (computer-fitted to a theoretical model) displayed two apparent pK values, of approx. 3.8 and 7.3. The temperature dependence of these pK values yielded heats of ionization of 3 and 0 kcal/mol from Van't Hoff plots for the lower and higher pK values, respectively. Reaction of alpha-L-fucosidase with 1-ethyl-3-(3-dimethylaminopropyl)carbodiimide and sodium p-(hydroxymercuri)benzoate resulted in complete inactivation of the enzyme. Other nonspecific inactivators had little or no effect on enzyme activity. These results suggest two carboxyl groups whose ionization state is important to activity, a non-active-site cysteine residue important to activity, and at least one active-site carboxyl group.     

Influence of flavin analogue structure on the catalytic activities and flavinylation reactions of recombinant human liver monoamine oxidases A and B.

Two riboflavin-deficient (rib5) Saccharomyces cerevisiae expression systems have been developed to investigate the influence of riboflavin structural alterations on the covalent flavinylation reaction and activity of recombinant human liver monoamine oxidases A and B (MAO A and B). Nineteen different riboflavin analogues were tested with MAO A and nine with MAO B. MAO expression and flavinylation were determined immunochemically with antisera to MAO and an anti-flavin antisera. Expression levels of both MAO A and B are invariant with the presence or absence of riboflavin or riboflavin analogues in the growth medium. Flavin analogues with a variety of seven and eight substitutions are found to be covalently incorporated and to confer catalytic activity. The selectivities of MAO A and MAO B for flavin analogue incorporation are found to be similar, although 8alpha-methylation of the flavin resulted in a higher level of catalytic activity for MAO B than for MAO A. N(3)-Methylriboflavin and 8-nor-8-aminoriboflavin are not covalently bound as they are not converted to their respective FAD forms by yeast. 5-Carba-5-deazaflavin and 7,8-nor-7-chlororiboflavin are not covalently incorporated into MAO A and do not support catalytic activity. A flavin peptide was isolated from MAO A containing 7-nor-7-bromo-FAD and was demonstrated to be covalently attached to Cys-406 by an 8alpha-S-thioether linkage by sequence analysis and by matrix-assisted laser desorption ionization time of flight mass spectroscopy. MAO A partially purified from yeast grown on 8-nor-8-chlororiboflavin exhibited an absorption spectrum indicating the covalent flavin is an 8-nor-8-S-thioflavin, suggesting a nucleophilic displacement mechanism that supports the quinone-methide mechanism previously suggested as a general mechanism for covalent flavin attachment.