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H Michel

Publications and source records attributed to H Michel.

At least 37 records · Page 2Linked to original sources
PubMed2000

Uphill electron transfer in the tetraheme cytochrome subunit of the Rhodopseudomonas viridis photosynthetic reaction center: evidence from site-directed mutagenesis.

The cytochrome (cyt) subunit of the photosynthetic reaction center from Rhodopseudomonas viridis contains four heme groups in a linear arrangement in the spatial order heme1, heme2, heme4, and heme3. Heme3 is the direct electron donor to the photooxidized primary electron donor (special pair, P(+)). This heme has the highest redox potential (E(m)) among the hemes in the cyt subunit. The E(m) of heme3 has been specifically lowered by site-directed mutagenesis in which the Arg residue at the position of 264 of the cyt was replaced by Lys. The mutation decreases the E(m) of heme3 from +380 to +270 mV, i.e., below that of heme2 (+320 mV). In addition, a blue shift of the alpha-band was found to accompany the mutation. The assignment of the lowered E(m) and the shifted alpha-band to heme3 was confirmed by spectroscopic measurements on RC crystals. The structure of the mutant RC has been determined by X-ray crystallography. No remarkable differences were found in the structure apart from the mutated residue itself. The velocity of the electron transfer (ET) from the tetraheme cyt to P(+) was measured under several redox conditions by following the rereduction of P(+) at 1283 nm after a laser flash. Heme3 donates an electron to P(+) with t(1/2) = 105 ns, i.e., faster than in the wild-type reaction center (t(1/2) = 190 ns), as expected from the larger driving force. The main feature is that a phase with t(1/2) approximately 2 micros dominates when heme3 is oxidized but heme2 is reduced. We conclude that the ET from heme2 to heme3 has a t(1/2) of approximately 2 micros, i.e., the same as in the WT, despite the fact that the reaction is endergonic by 50 meV instead of exergonic by 60 meV. We propose that the reaction kinetics is limited by the very uphill ET from heme2 to heme4, the DeltaG degrees of which is about the same (+230 meV) in both cases. The interpretation is further supported by measurements of the activation energy (216 meV in the wild-type, 236 meV in the mutant) and by approximate calculations of ET rates. Altogether these results demonstrate that the ET from heme2 to heme3 is stepwise, starting with a first very endergonic step from heme2 to heme4.

Amino Acid Substitution↗
PubMed2000

The monoclonal antibody 1F6 identifies a pH-dependent conformational change in the hydrophilic NH(2) terminus of NhaA Na(+)/H(+) antiporter of Escherichia coli.

One of the most interesting properties of the NhaA Na(+)/H(+) antiporter of Escherichia coli is the strong regulation of its activity by pH. This regulation is accompanied by a conformational change that can be probed by digestion with trypsin and involves the hydrophilic loop connecting the transmembrane helices VIII-IX. In the present work we show that a monoclonal antibody (mAb), 1F6, recognizes yet another domain of NhaA in a pH-dependent manner. This antibody binds NhaA at pH 8.5 but not at pH 4.5, whereas two other mAbs bind to NhaA independently of pH. The epitope of mAb 1F6 was located at the NH(2) terminus of NhaA by probing proteolytic fragments in Western blot analysis and amino acid sequencing. The antibody bound to the peptide HLHRFFSS, starting at the third amino acid of NhaA. A synthetic peptide with this sequence was shown to bind mAb 1F6 both at acidic and alkaline pH suggesting that this peptide is accessible to mAb 1F6 in the native protein only at alkaline pH. Although slightly shifted to acidic pH, the pH profile of the binding of mAb 1F6 to the antiporter is similar to that of both the Na(+)/H(+) antiporter activity as well as to its sensitivity to trypsin. We thus suggest that these pH profiles reflect a pH-dependent conformational change, which leads to activation of the antiporter. Indeed, a replacement of Gly-338 by Ser (G338S), which alleviates the pH dependence of both the NhaA activity as well as its sensitivity to trypsin, affects in a similar pattern the binding of mAb 1F6 to NhaA. Furthermore, the binding site of mAb 1F6 is involved in the functioning of the antiporter as follows: a double Cys replacement H3C/H5C causes an acidic shift by half a pH unit in the pH dependence of the antiporter; N-ethylmaleimide, which does not inhibit the wild-type protein, inhibits H3C/H5C antiporter to an extent similar to that exerted by mAb 1F6.

Amino Acid Sequence↗
PubMed2000

Engineering of a proteolytically stable human beta 2-adrenergic receptor/maltose-binding protein fusion and production of the chimeric protein in Escherichia coli and baculovirus-infected insect cells.

The hydrophobic human beta 2 adrenergic receptor was produced in fusion to the hydrophilic maltose-binding protein (MalE) in Escherichia coli. Photoaffinity labeling with the adrenergic ligand [125I]cyanopindolole-diazirine indicated that the majority of the protein was proteolyzed in the intergenic region between the fusion partners after production in E. coli. The simple and fast genetics of the bacterium enabled us to engineer a linker with an increased proteolytic stability. The fusion protein produced in E. coli was fully functional with respect to binding of adrenergic ligands and coupling to stimulatory GTP-binding protein. The production level with 3 pmol receptor fusion protein per mg membrane protein in a crude membrane preparation was significantly higher than those reported for other beta 2 adrenergic receptor constructs in E. coli. After solubilization with dodecanoyl sucrose, the fusion protein was purified to near homogeneity by affinity chromatography on immobilized Ni2+ ions (binding to a C-terminal His6-tag) and on crosslinked amylose (binding to the MalE). In order to achieve higher production levels, the fusion protein preceded by an insect signal peptide was produced in baculovirus-infected insect cells. As expected, the production level with about 17 pmol receptor per mg membrane protein was higher in the insect cells than in E. coli. The receptor fusion protein produced in the insect cells bound adrenergic ligands and activated heterotrimeric GTP-binding proteins with biochemical properties comparable to that of the unfused receptor.

ATP-Binding Cassette Transporters↗
PubMed2000

Functional properties of the heme propionates in cytochrome c oxidase from Paracoccus denitrificans. Evidence from FTIR difference spectroscopy and site-directed mutagenesis.

By specific (13)C labeling of the heme propionates, four bands in the reduced-minus-oxidized FTIR difference spectrum of cytochrome c oxidase from Paracoccus denitrificans have been assigned to the heme propionates [Behr, J., Hellwig, P., Mäntele, W., and Michel, H. (1998) Biochemistry 37, 7400-7406]. To attribute these signals to the individual propionates, we have constructed seven cytochrome coxidase variants using site-directed mutagenesis of subunit I. The mutant enzymes W87Y, W87F, W164F, H403A, Y406F, R473K, and R474K were characterized by measurement of enzymatic turnover, proton pumping activity, and Vis and FTIR spectroscopy. Whereas the mutant enzymes W164F and Y406F were found to be structurally altered, the other cytochrome c oxidase variants were suitable for band assignment in the infrared. Reduced-minus-oxidized FTIR difference spectra of the mutant enzymes were used to identify the ring D propionate of heme a as a likely proton acceptor upon reduction of cytochromic oxidase. The ring D propionate of heme a(3) might undergo conformational changes or, less likely, act as a proton donor.

Amino Acid Substitution↗
PubMed2000

Structure of the soluble domain of cytochrome c(552) from Paracoccus denitrificans in the oxidized and reduced states.

The crystal structure of the soluble domain of the membrane bound cytochrome c(552) (cytochrome c(552)') from Paracoccus denitrificans was determined using the multiwavelength anomalous diffraction technique and refined at 1.5 A resolution for the oxidized and at 1. 4 A for the reduced state. This is the first high-resolution crystal structure of a cytochrome c at low ionic strength in both redox states. The atomic model allowed for a detailed assessment of the structural properties including the secondary structure, the heme geometry and interactions, and the redox-coupled structural changes. In general, the structure has the same features as that of known eukaryotic cytochromes c. However, the surface properties are very different. Cytochrome c(552)' has a large strongly negatively charged surface part and a smaller positively charged area around the solvent-exposed heme atoms. One of the internal water molecules conserved in all structures of eukaryotic cytochromes c is also present in this bacterial cytochrome c. It contributes to the interactions between the side-chain of Arg36 and the heme propionate connected to pyrrole ring A. Reduction of the oxidized crystals does not influence the conformation of cytochrome c(552)' in contrast to eukaryotic cytochromes c. The oxidized cytochrome c(552)', especially the region of amino acid residues 40 to 56, appears to be more flexible than the reduced one.

Crystallography, X-Ray↗
PubMed2000

Structural investigations of the CuA centre of nitrous oxide reductase from Pseudomonas stutzeri by site-directed mutagenesis and X-ray absorption spectroscopy.

Nitrous oxide reductase is the terminal component of a respiratory chain that utilizes N2O in lieu of oxygen. It is a homodimer carrying in each subunit the electron transfer site, CuA, and the substrate-reducing catalytic centre, CuZ. Spectroscopic data have provided robust evidence for CuA as a binuclear, mixed-valence metal site. To provide further structural information on the CuA centre of N2O reductase, site directed mutagenesis and Cu K-edge X-ray absorption spectroscopic investigation have been undertaken. Candidate amino acids as ligands for the CuA centre of the enzyme from Pseudomonas stutzeri ATCC14405 were substituted by evolutionary conserved residues or amino acids similar to the wild-type residues. The mutations identified the amino acids His583, Cys618, Cys622 and Met629 as ligands of Cu1, and Cys618, Cys622 and His626 as the minimal set of ligands for Cu2 of the CuA centre. Other amino acid substitutions indicated His494 as a likely ligand of CuZ, and an indirect role for Asp580, compatible with a docking function for the electron donor. Cu binding and spectroscopic properties of recombinant N2O reductase proteins point at intersubunit or interdomain interaction of CuA and CuZ. Cu K-edge X-ray absorption spectra have been recorded to investigate the local environment of the Cu centres in N2O reductase. Cu K-edge Extended X-ray Absorption Fine Structure (EXAFS) for binuclear Cu chemical systems show clear evidence for Cu backscattering at approximately 2.5 A. The Cu K-edge EXAFS of the CuA centre of N2O reductase is very similar to that of the CuA centre of cytochrome c oxidase and the optimum simulation of the experimental data involves backscattering from a histidine group with Cu-N of 1.92 A, two sulfur atoms at 2.24 A and a Cu atom at 2. 43 A, and allows for the presence of a further light atom (oxygen or nitrogen) at 2.05 A. The interpretation of the CuA EXAFS is in line with ligands assigned by site-directed mutagenesis. By a difference spectrum approach, using the Cu K-edge EXAFS of the holoenzyme and that of the CuA-only form, histidine was identified as a major contributor to the backscattering. A structural model for the CuA centre of N2O reductase has been generated on the basis of the atomic coordinates for the homologous domain of cytochrome c oxidase and incorporating our current results and previous spectroscopic data.

Amino Acid Sequence↗
PubMed2000

The antitumoral effect of the long-acting somatostatin analog lanreotide in neuroendocrine tumors.

OBJECTIVE: Somatostatin analogs are the first-line drugs for controlling hormone-mediated symptoms of carcinoid tumors. Prospective and retrospective studies have suggested that somatostatin analogs also have antiproliferative activity. The octapeptide lanreotide is available in sustained-release form, obviating the need for daily injections. METHODS: A total of 46 patients were enrolled in this open, prospective, phase II trial. They received lanreotide 30 mg i.m. every 14 days for 6 months when they had symptomatic carcinoid tumors, and lanreotide 30 mg i.m. every 10 days if they had nonsymptomatic tumors. Nonsymptomatic tumors were progressive before the start of the study. Tumor size was assessed every 3 months by means of computed tomography. The assessment was centralized and was made by an external panel. RESULTS: In all, 30 patients had symptomatic neuroendocrine tumors and 16 had asymptomatic neuroendocrine tumors. Five patients in the group with symptomatic tumors and two in the group with nonsymptomatic tumors were considered not to be evaluable. The mean duration of treatment was 12 months in the group with symptomatic tumors and 13 months in the other group. Among the 39 evaluable patients, two objective responses were obtained, giving an objective response rate of 5% (one in the group with symptomatic tumors and one in the other group). Nineteen patients had no significant increase in their tumor size for a mean of 9.5 months. CONCLUSIONS: Lanreotide is safe and well tolerated in patients with carcinoid tumors. It seems to have both symptomatic and antitumoral effects in this setting.

Antineoplastic Agents↗
PubMed2000

The human ET(B) endothelin receptor heterologously produced in the methylotrophic yeast Pichia pastoris shows high-affinity binding and induction of stacked membranes.

The human endothelin B receptor (ET(B) receptor) was produced in the methylotrophic yeast Pichia pastoris under transcriptional control of the highly inducible alcohol oxidase 1 (AOX1) gene promoter. In the expression plasmids pPIC9KFlagET(B)Bio and pPIC9KFlag deltaGPET(B)Bio the ET(B) receptor coding region was fused in frame to the Saccharomyces cerevisiae alpha-factor prepropeptide and the FLAG-tag. In both constructs, the receptor was also fused to a biotinylation domain. Additionally, in pPIC9KFlag deltaGPET(B)Bio the putative N-glycosylation site and a protease site have been deleted by site directed mutagenesis. Crude membranes prepared from recombinant P. pastoris revealed specific and saturable binding of [125I]ET-1 with a K(D) of about 42 pM. Receptor levels of 60 pmol/mg and 35 pmol/mg for the Flag deltaGPET(B)Bio and the FlagET(B)Bio construct, respectively, were determined. The pharmacological profile for ET-1, ET-2 and ET-3 were as expected for a subtype B endothelin (ET) receptor. Immunoblot analysis showed an apparent molecular mass of 55 kDa for the Kex2-processed and about 74 kDa for the Kex2-unprocessed receptor. Contrary to the Flag deltaGPET(B)Bio construct, the FlagET(B)Bio construct was not correctly processed by the internal Kex2 endopeptidase. As was detected by ultrastructural analysis of recombinant yeast cells, high-level production of the receptor resulted in the formation of stacked membranes.

Amino Acid Sequence↗
PubMed1999

Mutation of Arg-54 strongly influences heme composition and rate and directionality of electron transfer in Paracoccus denitrificans cytochrome c oxidase.

The effect of a single site mutation of Arg-54 to methionine in Paracoccus denitrificans cytochrome c oxidase was studied using a combination of optical spectroscopy, electrochemical and rapid kinetics techniques, and time-resolved measurements of electrical membrane potential. The mutation resulted in a blue-shift of the heme a alpha-band by 15 nm and partial occupation of the low-spin heme site by heme O. Additionally, there was a marked decrease in the midpoint potential of the low-spin heme, resulting in slow reduction of this heme species. A stopped-flow investigation of the reaction with ferrocytochrome c yielded a kinetic difference spectrum resembling that of heme a(3). This observation, and the absence of transient absorbance changes at the corresponding wavelength of the low-spin heme, suggests that, in the mutant enzyme, electron transfer from Cu(A) to the binuclear center may not occur via heme a but that instead direct electron transfer to the high-spin heme is the dominating process. This was supported by charge translocation measurements where Deltapsi generation was completely inhibited in the presence of KCN. Our results thus provide an example for how the interplay between protein and cofactors can modulate the functional properties of the enzyme complex.

Arginine↗
PubMed1999

Identification of a hydrogen bond in the phe M197-->Tyr mutant reaction center of the photosynthetic purple bacterium Rhodobacter sphaeroides by X-ray crystallography and FTIR spectroscopy.

In bacterial reaction centers the charge separation process across the photosynthetic membrane is predominantly driven by the excited state of the bacteriochlorophyll dimer (D). An X-ray structure analysis of the Phe M197-->Tyr mutant reaction center from Rhodobacter sphaeroides at 2.7 A resolution suggests the formation of a hydrogen bond as postulated by Wachtveitl et al. [Biochemistry 32, 12875-12886, 1993] between the Tyr M197 hydroxy group and one of the 2a-acetyl carbonyls of D. In combination with electrochemically induced FTIR difference spectra showing a split band of the pi-conjugated 9-keto carbonyl of D, there is clear evidence for the existence of such a hydrogen bond.

Crystallography, X-Ray↗
PubMed1999

Structure of fumarate reductase from Wolinella succinogenes at 2.2 A resolution.

Fumarate reductase couples the reduction of fumarate to succinate to the oxidation of quinol to quinone, in a reaction opposite to that catalysed by the related complex II of the respiratory chain (succinate dehydrogenase). Here we describe the crystal structure at 2.2 A resolution of the three protein subunits containing fumarate reductase from the anaerobic bacterium Wolinella succinogenes. Subunit A contains the site of fumarate reduction and a covalently bound flavin adenine dinucleotide prosthetic group. Subunit B contains three iron-sulphur centres. The menaquinol-oxidizing subunit C consists of five membrane-spanning, primarily helical segments and binds two haem b molecules. On the basis of the structure, we propose a pathway of electron transfer from the dihaem cytochrome b to the site of fumarate reduction and a mechanism of fumarate reduction. The relative orientations of the soluble and membrane-embedded subunits of succinate:quinone oxidoreductases appear to be unique.

Cell Membrane↗
PubMed1999

The cytochrome c oxidase from Paracoccus denitrificans does not change the metal center ligation upon reduction.

Cytochrome c oxidase catalyzes the reduction of oxygen to water. This process is accompanied by the vectorial transport of protons across the mitochondrial or bacterial membrane ("proton pumping"). The mechanism of proton pumping is still a matter of debate. Many proposed mechanisms require structural changes during the reaction cycle of cytochrome c oxidase. Therefore, the structure of the cytochrome c oxidase was determined in the completely oxidized and in the completely reduced states at a temperature of 100 K. No ligand exchanges or other major structural changes upon reduction of the cytochrome c oxidase from Paracoccus denitrificans were observed. The three histidine Cu(B) ligands are well defined in the oxidized and in the reduced states. These results are hardly compatible with the "histidine cycle" mechanisms formulated previously.

Crystallography, X-Ray↗
PubMed1999

Cytochrome c oxidase: catalytic cycle and mechanisms of proton pumping--a discussion.

Cytochrome c oxidase catalyzes the reduction of molecular oxygen to water, a process in which four electrons, four protons, and one molecule of oxygen are consumed. The reaction is coupled to the pumping of four additional protons across the membrane. According to the currently accepted concept, the pumping of all four protons occurs after the binding of oxygen to the reduced enzyme and is exclusively coupled to the last two electron transfer steps. A careful analysis of the existing data shows that there is no experimental evidence for this paradigm. It is more likely that only three protons are pumped during the second half of the catalytic cycle of cytochrome c oxidase after the reaction with oxygen. In this article a variant of a recent mechanistic model of proton pumping by electrostatic repulsion is discussed. It is based on the electroneutrality principle in a way that in the catalytic cycle each electron transfer to the membrane-embedded electron acceptors is charge-compensated by uptake of one proton. The mechanism takes into account the findings with mutant cytochrome c oxidases and explains the results of many recent experiments, including the effects of hydrogen peroxide.

Catalysis↗
PubMed1999

In Rhodobacter sphaeroides reaction centers, mutation of proline L209 to aromatic residues in the vicinity of a water channel alters the dynamic coupling between electron and proton transfer processes.

The X-ray crystallographic structure of the photosynthetic reaction center from Rhodobacter sphaeroides obtained at high resolution has revealed a number of internal water molecules (Ermler, U., Fritzsch, G., Buchanan, S. K., and Michel, H. (1994) Structure 2, 925-936; Stowell, M. H. B., McPhillips, T. M., Rees, D. C., Soltis, S. M., Abresch, E., and Feher, G. (1997) Science 276, 812-816). Some of them are organized into distinct hydrogen-bonded water chains that connect Q(B) (the terminal quinone electron acceptor of the reaction center) to the aqueous phase. To investigate the role of the water chains in the proton conduction process, proline L209, located immediately adjacent to a water chain, was mutated to the following residues: F, Y, W, E, and T. We have first analyzed the effects of the mutations on the kinetic and thermodynamic properties of the rate constants of the second electron transfer (k(AB)(2)) and of the coupled proton uptake (k(H)+) at the second flash. In all aromatic mutants, k(AB)(2) and k(H)+ are notably and concomitantly decreased compared to the wild-type, while no effect is observed in the other mutants. The temperature dependence of these rates shows activation energy values (DeltaH) similar for the proton and electron-transfer processes in the wild-type and in most of the mutants, except for the L209PW and L209PF mutants. The analysis of the enthalpy factors related to the electron and proton-transfer processes in the L209PF and the L209PW mutants allows to distinguish the respective effects of the mutations for both transfer reactions. It is noteworthy that in the aromatic mutants a substantial increase of the free energies of activation is observed (DeltaG(L209PY) < DeltaG(L209PF) < DeltaG(L209PW)) for both proton and electron-transfer reactions, while in the other mutants, DeltaG is not affected. The salt concentration dependence of k(AB)(2) shows, in the L209PF and L209PW mutants, a higher screening of the protein surface potential experienced by Q(B). Our data suggest that residues F and W in position L209 increase the polarizability of the internal water molecules and polar residues by altering the organization of the hydrogen-bond network. We have also analyzed the rates of the first electron-transfer reaction (k(AB)(1)), in the 100 micros time domain. These kinetics have previously been shown to reflect protein relaxation events possibly including proton uptake events (Tiede, D. M., Vazquez, J., Cordova, J., and Marone, P. M. (1996) Biochemistry 35, 10763-10775). Interestingly, in the L209PF and L209PW mutants, k(AB)(1) is notably decreased in comparison to the wild type and the other mutants, in a similar way as k(AB)(2) and k(H)+. Our data imply that the dynamic organization of this web is tightly coupled to the electron transfer process that is kinetically limited by protonation events and/or conformational rearrangements within the protein.

Amino Acid Substitution↗
PubMed1999

Mutations in the Ca2+ binding site of the Paracoccus denitrificans cytochrome c oxidase.

Recent structure determinations suggested a new binding site for a non-redox active metal ion in subunit I of cytochrome c oxidase both of mitochondrial and of bacterial origin. We analyzed the relevant metal composition of the bovine and the Paracoccus denitrificans enzyme and of bacterial site-directed mutants in several residues presumably liganding this ion. Unlike the mitochondrial enzyme where a low, substoichiometric content of Ca2+ was found, the bacterial wild-type (WT) oxidase showed a stoichiometry of one Ca per enzyme monomer. Mutants in Asp-477 (in immediate vicinity of this site) were clearly diminished in their Ca content and the isolated mutant enzyme revealed a spectral shift in the heme a visible absorption upon Ca addition, which was reversed by Na ions. This spectral behavior, largely comparable to that of the mitochondrial enzyme, was not observed for the bacterial WT oxidase. Further structure refinement revealed a tightly bound water molecule as an additional Ca2+ ligand.

Animals↗
PubMed1999

Direct evidence for a tyrosine radical in the reaction of cytochrome c oxidase with hydrogen peroxide.

Cytochrome c oxidase (COX) catalyzes the reduction of oxygen to water, a process which is accompanied by the pumping of four protons across the membrane. Elucidation of the structures of intermediates in these processes is crucial for understanding the mechanism of oxygen reduction. In the work presented here, the reaction of H(2)O(2) with the fully oxidized protein at pH 6.0 has been investigated with electron paramagnetic resonance (EPR) spectroscopy. The results reveal an EPR signal with partially resolved hyperfine structure typical of an organic radical. The yield of this radical based on comparison with other paramagnetic centers in COX was approximately 20%. Recent crystallographic data have shown that one of the Cu(B) ligands, His 276 (in the bacterial case), is cross-linked to Tyr 280 and that this cross-linked tyrosine is ideally positioned to participate in dioxygen activation. Here selectively deuterated tyrosine has been incorporated into the protein, and a drastic change in the line shape of the EPR signal observed above has been detected. This would suggest that the observed EPR signal does indeed arise from a tyrosine radical species. It would seem also quite possible that this radical is an intermediate in the mechanism of oxygen reduction.

Copper↗
PubMed1999

Time-resolved FT-IR studies on the CO adduct of Paracoccus denitrificans cytochrome c oxidase: comparison of the fully reduced and the mixed valence form.

The rebinding of CO to cytochrome c oxidase from Paracoccus denitrificans in the fully reduced and in the half-reduced (mixed valence) form as a function of temperature was investigated using time-resolved rapid-scan FT-IR spectroscopy in the mid-IR (1200-2100 cm-1). For the fully reduced enzyme, rebinding was complete in approximately 2 s at 268 K and showed a biphasic reaction. At 84 K, nonreversible transfer of CO from heme a3 to CuB was observed. Both photolysis at 84 K and photolysis at 268 K result in FT-IR difference spectra which show similarities in the amide I, amide II, and heme modes. Both processes, however, differ in spectral features characteristic for amino acid side chain modes and may thus be indicative for the motional constraint of CO at low temperature. Rebinding of photodissociated CO for the mixed-valence enzyme at 268 K is also biphasic, but much slower as compared to the fully reduced enzyme. FT-IR difference spectra show band features similar to those for the fully reduced enzyme. Additional strong bands in the amide I and amide II range indicate local conformational changes induced by electron and coupled proton transfer. These signals disappear when the temperature is lowered to 84 K. At 268 K, a difference signal at 1746 cm-1 is observed which is shifted by 6 cm-1 to 1740 cm-1 in 2H2O. The absence of this signal for the mutant Glu 278 Gln allows assignment to the COOH stretching mode of Glu 278, and indicates changes of the conformation, proton position, or protonation of this residue upon electron transfer.

Carbon Monoxide↗