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PubMed1995

Roles of dipolar effects and local charge in the ionic strength dependence of redox reactions between c-type cytochromes.

Redox reactions between different c-type cytochromes were monitored by stopped-flow spectroscopy. Second-order rate constants were determined at different ionic strengths for the reactions of Paracoccus denitrificans cytochrome c-551i with its physiologic redox partner cytochrome c-550, and with the nonphysiologic partner horse heart cytochrome c. The latter two cytochromes are structurally quite similar and exhibit identical redox potentials but bear net charges of -7 and +7, respectively. Despite these opposite overall charges, the ionic strength dependencies for the reaction of each with the acidic cytochrome c-551i were very similar. The observed decrease in reaction rate with increasing ionic strength that was observed with cytochromes c-550 and c-551i, the latter of which bears a net charge of -20, cannot be explained simply on the basis of monopole-monopole interactions. These data were analyzed by two different methods: one which treats proteins as both monopoles and dipoles and considers the net charge; and another which neglects dipolar effects and considers only the local charge of the reactive site of the protein rather than net charge. The applicability of each method to the analysis of these data and to protein electrostatic interactions in general is discussed.

Bacterial Proteins↗
PubMed2000

Proton-coupled structural changes upon binding of carbon monoxide to cytochrome cd1: a combined flash photolysis and X-ray crystallography study.

We have investigated dynamic events after flash photolysis of CO from reduced cytochrome cd(1) nitrite reductase (NiR) from Paracoccus pantotrophus (formerly Thiosphaera pantotropha). Upon pulsed illumination of the cytochrome cd(1)-CO complex, at 460 nm, a rapid (<50 ns) absorbance change, attributed to dissociation of CO, was observed. This was followed by a biphasic rearrangement with rate constants of 1.7 x 10(4) and 2.5 x 10(3) s(-1) at pH 8.0. Both parts of the biphasic rearrangement phases displayed the same kinetic difference spectrum in the region of 400-660 nm. The slower of the two processes was accompanied by proton uptake from solution (0.5 proton per active site at pH 7.5-8.5). After photodissociation, the CO ligand recombined at a rate of 12 s(-1) (at 1 mM CO and pH 8.0), accompanied by proton release. The crystal structure of reduced cytochrome cd(1) in complex with CO was determined to a resolution of 1.57 A. The structure shows that CO binds to the iron of the d(1) heme in the active site. The ligation of the c heme is unchanged in the complex. A comparison of the structures of the reduced, unligated NiR and the NiR-CO complex indicates changes in the puckering of the d(1) heme as well as rearrangements in the hydrogen-bonding network and solvent organization in the substrate binding pocket at the d(1) heme. Since the CO ligand binds to heme d(1) and there are structural changes in the d(1) pocket upon CO binding, it is likely that the proton uptake or release observed after flash-induced CO dissociation is due to changes of the protonation state of groups in the active site. Such proton-coupled structural changes associated with ligand binding are likely to affect the redox potential of heme d(1) and may regulate the internal electron transfer from heme c to heme d(1).

Binding Sites↗
PubMed1995

Subunit III of cytochrome c oxidase influences the conformation of subunits I and II: an infrared study.

The secondary structure of wild-type Paracoccus denitrificans cytochrome c oxidase obtained by decomposition of the infrared amide I band contains 44% alpha-helix, 18% beta-sheet, 14% beta-turns, 18% loops, and 6% nonordered segments. The mutant lacking subunit III presents a small but significant increase (from 18% to 24%) in the percentage of loops and slight differences in the other components. Using band/area ratios and tyrosine side chain absorption as an inner standard, it is shown that in the absence of subunit III the structure of subunits I and II is altered although no changes in their alpha-helix or beta-sheet content are observed. In the bacterial oxidase, thermal infrared studies show a complex denaturation pattern characterized by the presence of a partially denatured intermediate state. Of the seven predicted subunit III alpha-helices, only four are resistant toward the thermal challenge and behave as expected for typical transmembrane helices. The observation that the absence of subunit III influences the conformation of loop regions in the two other subunits suggests that part of the interaction surface between subunit III and the catalytic subunits might be located outside the lipid bilayer.

Electron Transport Complex IV↗
PubMed1995

Involvement of cytochrome c oxidase subunit III in energy coupling.

The role of the conserved acidic residues of subunit III of cytochrome c oxidase (COIII) in energy transduction was investigated. Using a COIII deletion mutant of Paracoccus denitrificans, complemented with a plasmid expressing either the wild type (wt) COIII gene or site-directed mutants of the COIII gene, we measured cytochrome c oxidase-dependent ATP synthesis, respiration, and membrane potential. Cytochrome c oxidase-dependent ATP synthesis was attenuated in nonacidic mutants of either Glu98 (E98A and E98Q), or Asp259 (D259A) but not in the acidic mutant E98D. The rates of respiration in the energy conversion-defective mutants were as high as or higher than that in the wt. The cytochrome c oxidase-induced increment of membrane potential in the nonacidic mutants was similar to or higher than that in the wt. In contrast, when succinate-driven ATP synthesis was mediated solely by ubiquinol oxidase (e.g., in the presence of myxothiazol), the rates of ATP synthesis in the nonacidic mutants were higher than that in the wt. Moreover, myxothiazol, which inhibited succinate respiration as well as ATP synthesis in wt and E98D, stimulated ATP synthesis, while inhibiting succinate respiration, in the nonacidic mutants. These results indicate that the attenuation of energy conversion in these mutants is limited to cytochrome c oxidase and thus suggest that subunit III plays a role in energy conversion by cytochrome c oxidase.

Adenosine Triphosphate↗
PubMed1993

Deuterium kinetic isotope effect and stopped-flow kinetic studies of the quinoprotein methylamine dehydrogenase.

Stopped-flow kinetic studies of the reductive half-reaction of methylamine dehydrogenase from Paracoccus denitrificans yielded kinetic constants for the reversible formation of the imine intermediate formed between the substrate and the tryptophan tryptophylquinone (TTQ) prosthetic group and for the hydrogen abstraction step which occurs concomitantly with TTQ reduction. When CD3NH2 was used as a substrate, deuterium kinetic isotope effects of 4.2 and 3.8, respectively, were measured for the rate constants that correspond to the formation and dissociation of the iminoquinone intermediate. A deuterium kinetic isotope effect of 17.2 was measured for the hydrogen abstraction step. The maximum deuterium kinetic isotope effect which was measured in steady-state kinetic experiments was 3.0. These data are discussed in relation to the reaction mechanism of methylamine dehydrogenase and the similar large deuterium kinetic isotope effect for hydrogen abstraction which has been observed for another quinoprotein, plasma amine oxidase.

Deuterium↗
PubMed1993

A novel quinoprotein methanol dehydrogenase containing an additional 32-kilodalton peptide purified from Acetobacter methanolicus: identification of the peptide as a MoxJ product.

Acetobacter methanolicus is a unique acetic acid bacterium which has a methanol oxidase respiratory chain in addition to an ethanol oxidase respiratory chain. In this study, two different forms of methanol dehydrogenase (type I and II MDHs) were purified from A. methanolicus grown on methanol. Type I MDH was more basic (pI of 8.0) and smaller (M(r) of 148K) than type II MDH (pI of 6.7 and M(r) of 177K). Type I MDH consisted of alpha and beta subunits of 62 and 10 kDa, which has the same alpha 2 beta 2 conformation as the enzymes purified so far. The type II MDH contained an additional peptide of 32 kDa, of which a single copy was estimated to bind to the alpha 2 beta 2-MDH judging from the whole molecular weight and the stoichiometry of each subunit determined in sodium dodecyl sulfate-high-performance gel filtration chromatography. Compared with type I MDH, type II MDH exhibited a lower enzyme activity, but the electron-transfer activity to cytochrome cL was much more resistant to the inhibition with NaCl or EDTA. The possibility could be excluded that type II MDH is an artificial complex of type I MDH with a 32-kDa peptide, since it was inducible with methanol and could be detected in the periplasm, as well as the other subunits. Furthermore, the N-terminal amino acid sequence of the 32-kDa peptide showed a high homology to that of the moxJ product deduced from the DNA sequence of Paracoccus denitrificans or Methylobacterium extorquens AM1.(ABSTRACT TRUNCATED AT 250 WORDS)

Acetobacter↗
PubMed2000

Proton translocation by cytochrome c oxidase can take place without the conserved glutamic acid in subunit I.

A glutamic acid residue in subunit I of the heme-copper oxidases is highly conserved and has been directly implicated in the O(2) reduction and proton-pumping mechanisms of these respiratory enzymes. Its mutation to residues other than aspartic acid dramatically inhibits activity, and proton translocation is lost. However, this glutamic acid is replaced by a nonacidic residue in some structurally distant members of the heme-copper oxidases, which have a tyrosine residue in the vicinity. Here, using cytochrome c oxidase from Paracoccus denitrificans, we show that replacement of the glutamic acid and a conserved glycine nearby lowers the catalytic activity to <0.1% of the wild-type value. But if, in addition, a phenylalanine that lies close in the structure is changed to tyrosine, the activity rises more than 100-fold and proton translocation is restored. Molecular dynamics simulations suggest that the tyrosine can support a transient array of water molecules that may be essential for proton transfer in the heme-copper oxidases. Surprisingly, the glutamic acid is thus not indispensable, which puts important constraints on the catalytic mechanism of these enzymes.

Amino Acid Sequence↗
PubMed1991

Characterization of the tryptophan-derived quinone cofactor of methylamine dehydrogenase by resonance Raman spectroscopy.

The resonance Raman (RR) spectrum of oxidized methylamine dehydrogenase (MADHOX) exhibits a set of C-H, C-C, C = C, and C = O vibrational modes between 900 and 1700 cm-1 that are characteristic of the quinone moiety of the tryptophan tryptophlyquinone (TTQ) cofactor. The close similarity of the RR spectra for MADHs from Paracoccus denitrificans (Pd), Thiobacillus versutus (Tv), and bacterium W3A1 proves that the same cofactor is present in all three proteins. The MADHs from Pd and Tv have a v(C = O) mode at approximately 1625 cm-1 that shifts approximately 20 cm-1 upon 18O substitution of one of the carbonyl oxygens and is assigned to the in-phase symmetric stretch of the two C = O groups. The semiquinone form of Pd MADH has its own characteristic RR spectrum with altered peak frequencies and intensities as well as a decrease in the total number of peaks. The hydroxide and ammonia adducts of MADHOX produce RR spectra similar to that of the semiquinone. The spectral changes in all three cases are interpreted as being due to reduced conjugation of the cofactor. The ammonia adduct is formulated as a carbinolamine, a likely intermediate in the enzymatic mechanism. In contrast, formation of the electron-transfer complex between amicyanin and MADHOX has no effect on the vibrational frequencies (and, hence, structure) of either the MADH quinone or the amicyanin blue copper site. The behavior of the TTQ cofactors of Pd and Tv MADHs are very similar to one another and somewhat different from W3A1 MADH, particularly with regard to adduct formation and ability to undergo isotope exchange with solvent. These differences are ascribed to the cofactor environments within the proteins rather than to the structure of the cofactor itself.

Ammonia↗
PubMed2000

Mutagenesis of three conserved Glu residues in a bacterial homologue of the ND1 subunit of complex I affects ubiquinone reduction kinetics but not inhibition by dicyclohexylcarbodiimide.

Steady-state kinetics of the H(+)-translocating NADH:ubiquinone reductase (complex I) were analyzed in membrane samples from bovine mitochondria and the soil bacterium Paracoccus denitrificans. In both enzymes the calculated K(m) values, in the membrane lipid phase, for four different ubiquinone analogues were in the millimolar range. Both the structure and size of the hydrophobic side chain of the acceptor affected its affinity for complex I. The ND1 subunit of bovine complex I is a mitochondrially encoded protein that binds the inhibitor dicyclohexylcarbodiimide (DCCD) covalently [Yagi and Hatefi (1988) J. Biol. Chem. 263, 16150-16155]. The NQO8 subunit of P. denitrificans complex I is a homologue of ND1, and within it three conserved Glu residues that could bind DCCD, E158, E212, and E247, were changed to either Asp or Gln and in the case of E212 also to Val. The DCCD sensitivity of the resulting mutants was, however, unaffected by the mutations. On the other hand, the ubiquinone reductase activity of the mutants was altered, and the mutations changed the interactions of complex I with short-chain ubiquinones. The implications of the results for the location of the ubiquinone reduction site in this enzyme are discussed.

Amino Acid Sequence↗
PubMed1992

Crystal structure of an electron-transfer complex between methylamine dehydrogenase and amicyanin.

The crystal structure of the complex between the quinoprotein methylamine dehydrogenase (MADH) and the type I blue copper protein amicyanin, both from Paracoccus denitrificans, has been determined at 2.5-A resolution using molecular replacement. The search model was MADH from Thiobacillus versutus. The amicyanin could be located in an averaged electron density difference map and the model improved by refinement and model building procedures. Nine beta-strands are observed within the amicyanin molecule. The copper atom is located between three antiparallel strands and is about 2.5 A below the protein surface. The major intermolecular interactions occur between amicyanin and the light subunit of MADH where the interface is largely hydrophobic. The copper atom of amicyanin and the redox cofactor of MADH are about 9.4 A apart. One of the copper ligands, His 95, lies between the two redox centers and may facilitate electron transfer between them.

Bacterial Proteins↗
PubMed1991

Inhibition by cyclopropylamine of the quinoprotein methylamine dehydrogenase is mechanism-based and causes covalent cross-linking of alpha and beta subunits.

Cyclopropylamine acted as a mechanism-based inhibitor of the quinoprotein methylamine dehydrogenase from Paracoccus denitrificans. The protein-bound quinone cofactor of this enzyme was rapidly reduced by addition of a stoichiometric amount of cyclopropylamine, but this compound did not serve as a substrate for the enzyme in the steady-state kinetic assay. Time-dependent inactivation of the enzyme by cyclopropylamine was observed only in the presence of a reoxidant. Saturation behavior was observed, and values of KI of 3.9 microM and K(inact) of 1.7 min-1 were determined. Enzyme inactivation was irreversible, as no restoration of activity was evident after gel filtration of methylamine dehydrogenase which had been incubated with cyclopropylamine in the presence of a reoxidant. The inactivated enzyme exhibited an altered absorption spectrum. Electrophoretic analysis of inactivated methylamine dehydrogenase indicated that covalent cross-linking of the alpha and beta subunits of this alpha 2 beta 2 oligomeric enzyme had occurred and that the quinone cofactor had been modified. A mechanism for this inhibition is proposed which is based upon the data presented and is consistent with the available structural information on methylamine dehydrogenase.

Binding Sites↗
PubMed2001

Intramolecular electron transfer from c heme to d1 heme in bacterial cytochrome cd1 nitrite reductase occurs over the same distances at very different rates depending on the source of the enzyme.

Intramolecular electron transfer over 12 A from heme c to heme d(1) was investigated in cytochrome cd(1) nitrite reductase from Pseudomonas aeruginosa, following reduction of the c heme by pulse radiolysis. The rate constant for the transfer is relatively slow, k = 3 s(-1). The present observations contrast with a corresponding rate of electron transfer, 1.4 x 10(3) s(-1), measured for cytochrome cd(1) from Paracoccus pantotrophus, though the relative positions of the two heme groups are the same in both enzymes. The rate of intramolecular electron transfer within the enzyme from P. aeruginosa was accelerated 10(4)-fold (1.4 x 10(4) s(-1)) by the binding of cyanide to the d(1) heme. A coordination change at the d(1) heme upon its reduction is suggested to be a major factor in determining the slow rate of electron transfer in the P. aeruginosa enzyme in the absence of cyanide.

Cytochrome c Group↗
PubMed2001

Catalytic protein film voltammetry from a respiratory nitrate reductase provides evidence for complex electrochemical modulation of enzyme activity.

The first step in the respiratory reduction of nitrate to dinitrogen in Paracoccus pantotrophus is catalyzed by the quinol-nitrate oxidoreductase NarGHI. This membrane-anchored protein directs electrons from quinol oxidation at the membrane anchor, NarI, to the site of nitrate reduction in the membrane extrinsic [Fe-S] cluster and Mo-bis-MGD containing dimer, NarGH. Liberated from the membrane, NarGH retains its nitrate reductase activity and forms films on graphite and gold electrodes within which direct and facile exchange of electrons between the electrode and the enzyme occurs. Protein film voltammetry has been used to define the catalytic behavior of NarGH in the potential domain and a complex pattern of reversible, nitrate concentration dependent modulation of activity has been resolved. At low nitrate concentrations the local maximum observed in the catalytic current-potential profile reveals how NarGH can catalyze nitrate reduction via two pathways having distinct specificity constants, k(obs)(cat)/K(obs)(M). Catalysis is directed to occur via one of the pathways by an electrochemical event within NarGH. On increasing the nitrate concentration, the local maximum in the catalytic current becomes less distinct, and the catalytic waveform adopts an increasingly sigmoidal form. A pattern of voltammetry similar to that observed during nitrate reduction is observed during reduction of the stereochemically distinct substrate chlorate. Centers whose change of oxidation state may define the novel catalytic voltammetry of NarGH have been identified by EPR-monitored potentiometric titrations and mechanisms by which the electrochemistry of Mo-bis-MGD or [Fe-S] clusters can account for the observed behavior are discussed.

Catalysis↗
PubMed1986

Electron-transfer reactions between flavodoxin semiquinone and c-type cytochromes: comparisons between various flavodoxins.

As an extension of previous work from this laboratory using Clostridium pasteurianum flavodoxin [Tollin, G., Cheddar, G., Watkins, J. A., Meyer, T. E., & Cusanovich, M. A. (1984) Biochemistry 23, 6345-6349], we have measured the rate constants as a function of ionic strength for electron transfer from the semiquinones of Clostridium MP, Anacystis nidulans, and Azotobacter vinelandii flavodoxins to the following oxidants: cytochrome c from tuna and horse, Paracoccus denitrificans cytochrome c2, Pseudomonas aeruginosa cytochrome c-551, and ferricyanide. The rate constants extrapolated to infinite ionic strength (k infinity) for the C. MP flavodoxin are all slightly smaller than for the C. pasteurianum flavodoxin, as would be predicted on the basis of the higher redox potential of the C. MP protein. This indicates that there is a close similarity between the surface topographies of the two proteins in the vicinity of the coenzyme binding site. Moreover, the electrostatic interactions between the two flavodoxins and the various oxidants are also approximately the same. These studies justify our previous use of the crystallographic structure of the C. MP flavodoxin to interpret kinetic results obtained with the structurally uncharacterized C. pasteurianum flavodoxin. Despite their lower redox potentials, both Anacystis and Azotobacter flavodoxins are appreciably less reactive toward all of these oxidants (as much as 2 orders of magnitude in some cases) than are the Clostridium flavodoxins.(ABSTRACT TRUNCATED AT 250 WORDS)

Animals↗
PubMed1989

Redox protein electron-transfer mechanisms: electrostatic interactions as a determinant of reaction site in c-type cytochromes.

The effect of ionic strength on the rate constant for electron transfer has been used to determine the magnitude and charge sign of the net electrostatic potential which exists in close proximity to the sites of electron transfer on various c-type cytochromes. The negatively charged ferricyanide ion preferentially reacts at the positively charged exposed heme edge region on the front side of horse cytochrome c and Paracoccus cytochrome c2. In contrast, at low ionic strength, the positively charged cobalt phenanthroline ion interacts with the negatively charged back side of cytochrome c2, and at high ionic strength at a positively charged site on the front side of the cytochrome. With horse cytochrome c, over the ionic strength range studied, cobalt phenanthroline reacts only at a positively charged site which is probably not at the heme edge. These inorganic oxidants do not react at the relatively uncharged exposed heme edge sites on Azotobacter cytochrome c5 and Pseudomonas cytochrome c-551, but rather at a negatively charged site which is away from the heme edge. The results demonstrate that at least two electron-transferring sites on a single cytochrome can be functional, depending on the redox reactant used and the ionic strength. Electrostatic interactions between charge distributions on the cytochrome surface and the other reactant, or interactions involving uncharged regions on the protein(s), are critical in determining the preferred sites of electron transfer and reaction rate constants. When unfavorable electrostatic effects occur at a site near the redox center, less optimal sites at a greater distance can become kinetically important.

Acinetobacter↗
PubMed1980

Kinetics of oxidation and reduction of high-potential iron-sulfur proteins with nonphysiological reactants.

We have investigated the kinetic and equilibrium oxidation-reduction properties of HIPIP's from four sources: Chromatium vinosum, Thiocapsa pfennigii, Rhodopseudomonas gelatinosa, and a paracoccus sp. These studies focused on the interaction of the various HIPIP's and the iron hexacyanides as a function of ionic strength, pH, and temperature. On the basis of the kinetic and equilibrium data obtained and the known structrual properties of the various HIPIP's a number of conclusions concerning the mechanism of electron transfer by HIPIP can be inferred. (1) The oxidation and reduction of HIPIP by the iron hexacyanides show no kinetic complexity due to heterogeneity or changes in rate-limiting step. (2) The kinetic and equilibrium studies (oxidation-reduction potential) are in good agreement, although a rapid binding equilibrium between HIPIP and the iron hexacyanides is possible. (3) The kinetics of oxidation and reduction of the various HIPIP's studied do not correlate with their oxidation-reduction potentials. (4) The electrostatic interactions between a particular HIPIP and the iron hexacyanides appear to be influenced by the charge on the iron-sulfur cluster with specific amino acid side chains playing a significant but limited role in the interactions leading to electron transfer. (5) The iron-sulfur cluster charge is apparently distributed on the surface of the HIPIP molecule through a network of hydrogen bonds. (6) On the basis of an analysis of the known primary and tertiary structure of the HIPIP's studied, it is concluded that peptide backbone carbonyls and amino acid side chains for the amino acid sequence positions 46-49, 63-65, 78-82, 31-34, and 16-17 define the interaction site of HIPIP and the iron hexacyanides irrespective of the HIPIP source. This is a structural region at which the iron-sulfur cluster is near to the protein surface and in this respect resembles the active site in mitochondrial cytochrome c.

Amino Acid Sequence↗
PubMed2001

Dynamics of nitric oxide in the active site of reduced cytochrome c oxidase aa3.

Nitric oxide (NO) is involved in the regulation of respiration by acting as a competitive ligand for molecular oxygen at the binuclear active site of cytochrome c oxidase. The dynamics of NO in and near this site are not well understood. We performed flash photolysis studies of NO from heme a3 in cytochrome c oxidase from Paracoccus denitrificans, using femtosecond transient absorption spectroscopy. The formation of the product state--the unliganded heme a3 ground state--occurs in a similar stepwise manner (period approximately 700 fs) as previously observed for carbon monoxide photolysis from this enzyme and interpreted in terms of ballistic ligand motions in the active site on the subpicosecond time scale [Liebl, U., Lipowski, G., Négrerie, M., Lambry, J.-C., Martin, J.-L., and Vos, M. H. (1999) Nature 401, 181-184]. A fraction (approximately 35% at very low NO concentrations) of the dissociated NO recombines with heme a3 in 200-300 ps. The presence of this recombination phase indicates that a transient bond to the second ligand-binding site, a copper atom (CuB), has a short lifetime or may not be formed. Increasing the NO concentration increases the recombination yield on the hundreds of picoseconds time scale. This effect, unprecedented for heme proteins, implies that, apart from the one NO molecule bound to heme a3, a second NO molecule can be accommodated in the active site, even at relatively low (submicromolar) concentrations. Models for NO accommodation in the active site, based on molecular dynamics energy minimizations are presented. Pathways for NO motion and their relevance for the regulation of respiration are discussed.

Binding Sites↗
PubMed2001

Carboxyl residues in the iron-sulfur protein are involved in the proton pumping activity of P. denitrificans bc(1) complex.

A study is presented on chemical modification of the three subunit Paracoccus denitrificans bc(1) complex. N-(Ethoxycarbonyl)-2-ethoxy-1,2-dihydroquinoline (EEDQ) treatment caused a loss of the proton pumping activity of liposome-reconstituted bc(1) complex. A similar effect, which is referred to as the decoupling effect, resulted upon reaction of N,N'-dicyclohexylcarbodiimide (DCCD) with the complex. Direct measurement of the binding of EEDQ to the complex subunits, performed in the presence of the fluorescent hydrophobic nucleophile 4'-[(aminoacetamido)methyl]fluorescein (AMF), showed that the iron-sulfur protein (ISP) and cytochrome c(1) were labeled by EEDQ, whereas cytochrome b was not. Tryptic digestion and sequencing analysis of the fluorescent fragment of the ISP revealed this to consist of a segment with six acidic residues, among which the highly conserved aspartate 160 is present. Analogous experiments on DCCD binding showed that all the three subunits of the complex were labeled. However, DCCD concentration dependence of carboxyl residue modification in the individual subunits and of proton pumping activity showed that the decrease of the H(+)/e(-) ratio correlated only with the modification of the ISP. Tryptic digestion of labeled ISP and sequencing analysis of the fluorescent fragment gave results superimposable upon those obtained with EEDQ. Chymotryptic digestion and sequencing analysis of the single fluorescent fragment of cytochrome b showed that this fragment contained glutamate 174 and aspartate 187. We conclude that, in the P. denitrificans bc(1) complex, carboxyl residues in cytochrome b do not appear to be critically involved in the proton pump mechanism of the complex.

Amino Acid Sequence↗