Activation of the cytochrome cd1 nitrite reductase from Paracoccus pantotrophus. Reaction of oxidized enzyme with substrate drives a ligand switch at heme c

Cytochromes cd(1) are dimeric bacterial nitrite reductases, which contain two hemes per monomer. On reduction of both hemes, the distal ligand of heme d(1) dissociates, creating a vacant coordination site accessible to substrate. Heme c, which transfers electrons from donor proteins into the active site, has histidine/methionine ligands except in the oxidized enzyme from Paracoccus pantotrophus where both ligands are histidine. During reduction of this enzyme, Tyr(25) dissociates from the distal side of heme d(1), and one heme c ligand is replaced by methionine. Activity is associated with histidine/methionine coordination at heme c, and it is believed that P. pantotrophus cytochrome cd(1) is unreactive toward substrate without reductive activation. However, we report here that the oxidized enzyme will react with nitrite to yield a novel species in which heme d(1) is EPR-silent. Magnetic circular dichroism studies indicate that heme d(1) is low-spin Fe(III) but EPR-silent as a result of spin coupling to a radical species formed during the reaction with nitrite. This reaction drives the switch to histidine/methionine ligation at Fe(III) heme c. Thus the enzyme is activated by exposure to its physiological substrate without the necessity of passing through the reduced state. This reactivity toward nitrite is also observed for oxidized cytochrome cd(1) from Pseudomonas stutzeri suggesting a more general involvement of the EPR-silent Fe(III) heme d(1) species in nitrite reduction.     

The peroxynitrite reductase activity of cytochrome c oxidase involves a two-electron redox reaction at the heme a(3)-Cu(B) site

Fully and partially reduced forms of isolated bovine cytochrome c oxidase undergo a two-electron oxidation-reduction process with added peroxynitrite, leading to catalytic oxidation of ferrocytochrome c to ferricytochrome c. The other major reaction product is nitrite ion, 86% of the added peroxynitrite being measurably converted to this species. The reaction is inhibited in the presence of cyanide, implicating the heme a(3)-Cu(B) binuclear pair as the active site. Moreover, provided peroxynitrite is not added to excess, the reductase activity of the enzyme toward this oxidant efficiently protects other protein and detergent molecules in vitro from nitration of tyrosine residues and oxidative damage. If the enzyme is exposed to approximately 10(2)-fold excesses of peroxynitrite, then significant irreversible loss of electron transfer activity results, and the heme a(3)-Cu(B) binuclear pair no longer undergo a characteristic carbon monoxide-driven reduction. The accompanying rather small changes in the observed electronic absorption spectrum are suggestive of a modification in the vicinity of one or both hemes but probably not to the cofactors themselves.     

Hexaheme nitrite reductase from Desulfovibrio desulfuricans. M?ssbauer and EPR characterization of the heme groups

M?ssbauer and EPR spectroscopy were used to characterize the heme prosthetic groups of the nitrite reductase isolated from Desulfovibrio desulfuricans (ATCC 27774), which is a membrane-bound multiheme cytochrome capable of catalyzing the 6-electron reduction of nitrite to ammonia. At pH 7.6, the as-isolated enzyme exhibited a complex EPR spectrum consisting of a low-spin ferric heme signal at g = 2.96, 2.28, and 1.50 plus several broad resonances indicative of spin-spin interactions among the heme groups. EPR redox titration studies revealed yet another low-spin ferric heme signal at g = 3.2 and 2.14 (the third g value was undetected) and the presence of a high-spin ferric heme. M?ssbauer measurements demonstrated further that this enzyme contained six distinct heme groups: one high-spin (S = 5/2) and five low-spin (S = 1/2) ferric hemes. Characteristic hyperfine parameters for all six hemes were obtained through a detailed analysis of the M?ssbauer spectra. D. desulfuricans nitrite reductase can be reduced by chemical reductants, such as dithionite or reduced methyl viologen, or by hydrogenase under hydrogen atmosphere. Addition of nitrite to the fully reduced enzyme reoxidized all five low-spin hemes to their ferric states. The high-spin heme, however, was found to complex NO, suggesting that the high-spin heme could be the substrate binding site and that NO could be an intermediate present in an enzyme-bound form.     

Electron transfer to the active site of the bacterial nitric oxide reductase is controlled by ligand binding to heme b₃

The active site of the bacterial nitric oxide reductase from Paracoccus denitrificans contains a dinuclear centre comprising heme b? and non heme iron (Fe(B)). These metal centres are shown to be at isopotential with midpoint reduction potentials of E(m) ≈ +80 mV. The midpoint reduction potentials of the other two metal centres in the enzyme, heme c and heme b, are greater than the dinuclear centre suggesting that they act as an electron receiving/storage module. Reduction of the low-spin heme b causes structural changes at the dinuclear centre which allow access to substrate molecules. In the presence of the substrate analogue, CO, the midpoint reduction potential of heme b? is raised to a region similar to that of heme c and heme b. This leads us to suggest that reduction of the electron transfer hemes leads to an opening of the active site which allows substrate to bind and in turn raises the reduction potential of the active site such that electrons are only delivered to the active site following substrate binding.     

High-Resolution Structural Analysis of a Novel Octaheme Cytochrome c Nitrite Reductase from the Haloalkaliphilic Bacterium Thioalkalivibrio nitratireducens

Bacterial pentaheme cytochrome c nitrite reductases (NrfAs) are key enzymes involved in the terminal step of dissimilatory nitrite reduction of the nitrogen cycle. Their structure and functions are well studied. Recently, a novel octaheme cytochrome c nitrite reductase (TvNiR) has been isolated from the haloalkaliphilic bacterium Thioalkalivibrio nitratireducens. Here we present high-resolution crystal structures of the apoenzyme and its complexes with the substrate (nitrite) and the inhibitor (azide). Both in the crystalline state and in solution, TvNiR exists as a stable hexamer containing 48 hemes—the largest number of hemes accommodated within one protein molecule known to date. The subunit of TvNiR consists of two domains. The N-terminal domain has a unique fold and contains three hemes. The catalytic C-terminal domain hosts the remaining five hemes, their arrangement, including the catalytic heme, being identical to that found in NrfAs. The complete set of eight hemes forms a spatial pattern characteristic of other multiheme proteins, including structurally characterized octaheme cytochromes. The catalytic machinery of TvNiR resembles that of NrfAs. It comprises the lysine residue at the proximal position of the catalytic heme, the catalytic triad of tyrosine, histidine, and arginine at the distal side, channels for the substrate and product transport with a characteristic gradient of electrostatic potential, and, finally, two conserved Ca²⁺-binding sites. However, TvNiR has a number of special structural features, including a covalent bond between the catalytic tyrosine and the adjacent cysteine and the unusual topography of the product channels that open into the void interior space of the protein hexamer. The role of these characteristic structural features in the catalysis by this enzyme is discussed.     

Proton and electron pathways in the bacterial nitric oxide reductase

Electron- and proton-transfer reactions in bacterial nitric oxide reductase (NOR) have been investigated by optical spectroscopy and electrometry. In liposomes, NOR does not show any generation of an electric potential during steady-state turnover. This electroneutrality implies that protons are taken up from the same side of the membrane as electrons during catalysis. Intramolecular electron redistribution after photolysis of the partially reduced CO-bound enzyme shows that the electron transfer in NOR has the same pathway as in the heme-copper oxidases. The electron is transferred from the acceptor site, heme c, via a low-spin heme b to the binuclear active site (heme b3/FeB). The electron-transfer rate between hemes c and b is (3 +/- 2) x 10(4) s(-1). The rate of electron transfer between hemes b and b3 is too fast to be resolved (>10(6) s(-1)). Only electron transfer between heme c and heme b is coupled to the generation of an electric potential. This implies that the topology of redox centers in NOR is comparable to that in the heme-copper cytochrome oxidases. The optical and electrometric measurements allow identification of the intermediate states formed during turnover of the fully reduced enzyme, as well as the associated proton and electron movement linked to the NO reduction. The first phase (k = 5 x 10(5) s(-1)) is electrically silent, and characterized by the disappearance of absorbance at 433 nm and the appearance of a broad peak at 410 nm. We assign this phase to the formation of a ferrous NO adduct of heme b3. NO binding is followed by a charge separation phase (k = 2.2 x 10(5) s(-1)). We suggest that the formation of this intermediate that is not linked to significant optical changes involves movement of charged side chains near the active site. The next step creates a negative potential with a rate constant of approximately 3 x 10(4) s(-1) and a weak optical signature. This is followed by an electrically silent phase with a rate constant of 5 x 10(3) s(-1) leading to the last intermediate of the first turnover (a rate constant of approximately 10(3) s(-1)). The fully reduced enzyme has four electrons, enough for two complete catalytic cycles. However, the protons for the second turnover must be taken from the bulk, resulting in the generation of a positive potential in two steps. The optical measurements also verify two phases in the oxidation of low-spin hemes. Based on these results, we present mechanistic models of NO reduction by NOR. The results can be explained with a trans mechanism rather than a cis model involving FeB. Additionally, the data open up the possibility that NOR employs a P450-type mechanism in which only heme b3 functions as the NO binding site during turnover.     

Substrate binding and activation in the active site of cytochrome <Emphasis Type="Italic">c</Emphasis> nitrite reductase: a density functional study

Cytochrome c nitrite reductase is a homodimeric enzyme, containing five covalently attached c-type hemes per subunit. Four of the heme irons are bishistidine-ligated, whereas the fifth, the active site of the protein, has an unusual lysine coordination and calcium site nearby. A fascinating feature of this enzyme is that the full six-electron reduction of the nitrite is achieved without release of any detectable reaction intermediate. Moreover, the enzyme is known to work over a wide pH range. Both findings suggest a unique flexibility of the active site in the complicated six-electron, seven-proton reduction process. In the present work, we employed density functional theory to study the energetics and kinetics of the initial stages of nitrite reduction. The possible role of second-sphere active-site amino acids as proton donors was investigated by taking different possible protonation states and geometrical conformations into account. It was found that the most probable proton donor is His₂₇₇, whose spatial orientation and fine-tuned acidity lead to energetically feasible, low-barrier protonation reactions. However, substrate protonation may also be accomplished by Arg₁₁₄. The calculated barriers for this pathway are only slightly higher than the experimentally determined value of 15.2 kcal/mol for the rate-limiting step. Hence, having proton-donating side chains of different acidity within the active site may increase the operational pH range of the enzyme. Interestingly, Tyr₂₁₈, which was proposed to play an important role in the overall mechanism, appears not to take part in the reaction during the initial stage.     

The isolation and characterization of cytochrome c nitrite reductase subunits (NrfA and NrfH) from Desulfovibrio desulfuricans ATCC 27774. Re-evaluation of the spectroscopic data and redox properties.

The cytochrome c nitrite reductase is isolated from the membranes of the sulfate-reducing bacterium Desulfovibrio desulfuricans ATCC 27774 as a heterooligomeric complex composed by two subunits (61 kDa and 19 kDa) containing c-type hemes, encoded by the genes nrfA and nrfH, respectively. The extracted complex has in average a 2NrfA:1NrfH composition. The separation of ccNiR subunits from one another is accomplished by gel filtration chromatography in the presence of SDS. The amino-acid sequence and biochemical subunits characterization show that NrfA contains five hemes and NrfH four hemes. These considerations enabled the revision of a vast amount of existing spectroscopic data on the NrfHA complex that was not originally well interpreted due to the lack of knowledge on the heme content and the oligomeric enzyme status. Based on EPR and M?ssbauer parameters and their correlation to structural information recently obtained from X-ray crystallography on the NrfA structure [Cunha, C.A., Macieira, S., Dias, J.M., Almeida, M.G., Gon?alves, L.M.L., Costa, C., Lampreia, J., Huber, R., Moura, J.J.G., Moura, I. & Rom?o, M. (2003) J. Biol. Chem. 278, 17455-17465], we propose the full assignment of midpoint reduction potentials values to the individual hemes. NrfA contains the high-spin catalytic site (-80 mV) as well as a quite unusual high reduction potential (+150 mV)/low-spin bis-His coordinated heme, considered to be the site where electrons enter. In addition, the reassessment of the spectroscopic data allowed the first partial spectroscopic characterization of the NrfH subunit. The four NrfH hemes are all in a low-spin state (S = 1/2). One of them has a gmax at 3.55, characteristic of bis-histidinyl iron ligands in a noncoplanar arrangement, and has a positive reduction potential.     

Binding and reduction of sulfite by cytochrome c nitrite reductase

Pentaheme cytochrome c nitrite reductase (ccNiR) catalyzes the six-electron reduction of nitrite to ammonia as the final step in the dissimilatory pathway of nitrate ammonification. It has also been shown to reduce sulfite to sulfide, thus forming the only known link between the biogeochemical cycles of nitrogen and of sulfur. We have found the sulfite reductase activity of ccNiR from Wolinella succinogenes to be significantly smaller than its nitrite reductase activity but still several times higher than the one described for dissimilatory, siroheme-containing sulfite reductases. To compare the sulfite reductase activity of ccNiR with our previous data on nitrite reduction, we determined the binding mode of sulfite to the catalytic heme center of ccNiR from W. succinogenes at a resolution of 1.7 A. Sulfite and nitrite both provide a pair of electrons to form the coordinative bond to the Fe(III) active site of the enzyme, and the oxygen atoms of sulfite are found to interact with the three active site protein residues conserved within the enzyme family. Furthermore, we have characterized the active site variant Y218F of ccNiR that exhibited an almost complete loss of nitrite reductase activity, while sulfite reduction remained unaffected. These data provide a first direct insight into the role of the first sphere of protein ligands at the active site in ccNiR catalysis.     

Tuning of functional heme reduction potentials in Shewanella fumarate reductases

The fumarate reductases from S. frigidimarina NCIMB400 and S. oneidensis MR-1 are soluble and monomeric enzymes located in the periplasm of these bacteria. These proteins display two redox active domains, one containing four c-type hemes and another containing FAD at the catalytic site. This arrangement of single-electron redox co-factors leading to multiple-electron active sites is widespread in respiratory enzymes. To investigate the properties that allow a chain of single-electron co-factors to sustain the activity of a multi-electron catalytic site, redox titrations followed by NMR and visible spectroscopies were applied to determine the microscopic thermodynamic parameters of the hemes. The results show that the redox behaviour of these fumarate reductases is similar and dominated by a strong interaction between hemes II and III. This interaction facilitates a sequential transfer of two electrons from the heme domain to FAD via heme IV.     

Cytochrome c nitrite reductase from Desulfovibrio desulfuricans ATCC 27774. The relevance of the two calcium sites in the structure of the catalytic subunit (NrfA)

The gene encoding cytochrome c nitrite reductase (NrfA) from Desulfovibrio desulfuricans ATCC 27774 was sequenced and the crystal structure of the enzyme was determined to 2.3-A resolution. In comparison with homologous structures, it presents structural differences mainly located at the regions surrounding the putative substrate inlet and product outlet, and includes a well defined second calcium site with octahedral geometry, coordinated to propionates of hemes 3 and 4, and caged by a loop non-existent in the previous structures. The highly negative electrostatic potential in the environment around hemes 3 and 4 suggests that the main role of this calcium ion may not be electrostatic but structural, namely in the stabilization of the conformation of the additional loop that cages it and influences the solvent accessibility of heme 4. The NrfA active site is similar to that of peroxidases with a nearby calcium site at the heme distal side nearly in the same location as occurs in the class II and class III peroxidases. This fact suggests that the calcium ion at the distal side of the active site in the NrfA enzymes may have a similar physiological role to that reported for the peroxidases.     

Nitric oxide reacts with the ferryl-oxo catalytic intermediate of the CuB-lacking cytochrome bd terminal oxidase

Cytochrome bd is a bacterial respiratory oxidase carrying three hemes but no copper. We show that nitric oxide (NO) reacts with the intermediate F of cytochrome bd from Azotobacter vinelandii: (i) with a 1:1 stoichiometry, (ii) rapidly (k=1.2 +/- 0.1 x 10(5)M(-1)s(-1) at 20 degrees C), and (iii) yielding the oxidized enzyme with nitrite bound to heme d at the active site. Unexpectedly, the NO reaction mechanism of this catalytic intermediate in the Cu(B)-lacking cytochrome bd appears similar to that of beef heart cytochrome c oxidase, where Cu(B) was proposed to play a key role.     

An octaheme c-type cytochrome from Shewanella oneidensis can reduce nitrite and hydroxylamine

A c-type cytochrome from Shewanella oneidensis MR-1, containing eight hemes, has been previously designated as an octaheme tetrathionate reductase (OTR). The structure of OTR revealed that the active site contains an unusual lysine-ligated heme, despite the presence of a CXXCH motif in the sequence that would predict histidine ligation. This lysine ligation has been previously observed only in the pentaheme nitrite reductases, suggesting that OTR may have a possible role in nitrite reduction. We have now shown that OTR is an efficient nitrite and hydroxylamine reductase and that ammonium ion is the product. These results indicate that OTR may have a role in the biological nitrogen cycle.     

Suicide inactivation of cytochrome c oxidase: catalytic turnover in the absence of subunit III alters the active site

The catalytic core of cytochrome c oxidase is composed of three subunits: I, II, and III. Subunit III is a highly hydrophobic membrane protein that contains no redox centers; its role in cytochrome oxidase function is not obvious. Here, subunit III has been removed from the three-subunit mitochondrial-like oxidase of Rhodobacter sphaeroides by detergent washing. The resulting two-subunit oxidase, subunit III (-), is highly active. Ligand-binding analyses and resonance Raman spectroscopy show that its heme a(3)-Cu(B) active site is normal. However, subunit III (-) spontaneously and irreversibly inactivates during O(2) reduction. At pH 7.5, its catalytic lifetime is only 2% that of the normal oxidase. This suicide inactivation event primarily alters the active site. Its ability to form specific O(2) reduction intermediates is lost, and CO binding experiments suggest that the access of O(2) to reduced heme a(3) is inhibited. Reduced heme a accumulates in response to a decrease in the redox potential of heme a(3); electron transfer between the hemes is inhibited. Ligand-binding experiments and resonance Raman analysis show that increased flexibility in the structure of the active site accompanies inactivation. Cu(B) is partially lost. It is proposed that suicide inactivation results from the dissociation of a ligand of Cu(B) and that subunit III functions to prevent suicide inactivation by maintaining the structural integrity of the Cu(B) center via long-range interactions.     

Electron transfer process in cytochrome bd-type ubiquinol oxidase from Escherichia coli revealed by pulse radiolysis

Cytochrome bd is a two-subunit ubiquinol oxidase in the aerobic respiratory chain of Escherichia coli and binds hemes b558, b595, and d as the redox metal centers. Taking advantage of spectroscopic properties of three hemes which exhibit distinct absorption peaks, we investigated electron transfer within the enzyme by the technique of pulse radiolysis. Reduction of the hemes in the air-oxidized, resting-state enzyme, where heme d exists in mainly an oxygenated form and partially an oxoferryl and a ferric low-spin forms, occurred in two phases. In the faster phase, radiolytically generated N-methylnicotinamide radicals simultaneously reduced the ferric hemes b558 and b595 with a second-order rate constant of 3 x 10(8) M-1 s-1, suggesting that a rapid equilibrium occurs for electron transfer between two b-type hemes long before 10 micros. In the slower phase, an intramolecular electron transfer from heme b to the oxoferryl and the ferric heme d occurred with the first-order rate constant of 4.2-5.6 x 10(2) s-1. In contrast, the oxygenated heme d did not exhibit significant spectral change. Reactions with the fully oxidized and hydrogen peroxide-treated forms demonstrated that the oxidation and/or ligation states of heme d do not affect the heme b reduction. The following intramolecular electron transfer transformed the ferric and oxoferryl forms of heme d to the ferrous and ferric forms, respectively, with the first-order rate constants of 3.4 x 10(3) and 5.9 x 10(2) s-1, respectively.     

Intramolecular electron transfer in cytochrome cd(1) nitrite reductase from Pseudomonas stutzeri; kinetics and thermodynamics

Cytochrome cd(1) nitrite reductase from Pseudomonas stutzeri catalyzes the one electron reduction of nitrite to nitric oxide. It is a homodimer, each monomer containing one heme-c and one heme-d(1), the former being the electron uptake site while the latter is the nitrite reduction site. Hence, internal electron transfer between these sites is an inherent element in the catalytic cycle of this enzyme. We have investigated the internal electron transfer reaction employing pulse radiolytically produced N-methyl nicotinamide radicals as reductant which reacts solely with the heme-c in an essentially diffusion controlled process. Following this initial step, the reduction equivalent is equilibrating between the c and d(1) heme sites in a unimolecular process (k=23 s(-1), 298 K, pH 7.0) and an equilibrium constant of 1.0. The temperature dependence of this internal electron transfer process has been determined over a 277-313 K temperature range and yielded both equilibrium standard enthalpy and entropy changes as well as activation parameters of the specific rate constants. The significance of these parameters obtained at low degree of reduction of the enzyme is discussed and compared with earlier studies on cd(1) nitrite reductases from other sources.     

Nitrite reductase activity of hemoglobin S (sickle) provides insight into contributions of heme redox potential versus ligand affinity

Hemoglobin A (HbA) is an allosterically regulated nitrite reductase that reduces nitrite to NO under physiological hypoxia. The efficiency of this reaction is modulated by two intrinsic and opposing properties: availability of unliganded ferrous hemes and R-state character of the hemoglobin tetramer. Nitrite is reduced by deoxygenated ferrous hemes, such that heme deoxygenation increases the rate of NO generation. However, heme reactivity with nitrite, represented by its bimolecular rate constant, is greatest when the tetramer is in the R quaternary state. The mechanism underlying the higher reactivity of R-state hemes remains elusive. It can be due to the lower heme redox potential of R-state ferrous hemes or could reflect the high ligand affinity geometry of R-state tetramers that facilitates nitrite binding. We evaluated the nitrite reductase activity of unpolymerized sickle hemoglobin (HbS), whose oxygen affinity and cooperativity profile are equal to those of HbA, but whose heme iron has a lower redox potential. We now report that HbS exhibits allosteric nitrite reductase activity with competing proton and redox Bohr effects. In addition, we found that solution phase HbS reduces nitrite to NO significantly faster than HbA, supporting the thesis that heme electronics (i.e. redox potential) contributes to the high reactivity of R-state deoxy-hemes with nitrite. From a pathophysiological standpoint, under conditions where HbS polymers form, the rate of nitrite reduction is reduced compared with HbA and solution-phase HbS, indicating that HbS polymers reduce nitrite more slowly.     

Role of a conserved glutamine residue in tuning the catalytic activity of Escherichia coli cytochrome c nitrite reductase

The pentaheme cytochrome c nitrite reductase (NrfA) of Escherichia coli is responsible for nitrite reduction during anaerobic respiration when nitrate is scarce. The NrfA active site consists of a hexacoordinate high-spin heme with a lysine ligand on the proximal side and water/hydroxide or substrate on the distal side. There are four further highly conserved active site residues including a glutamine (Q263) positioned 8 A from the heme iron for which the side chain, unusually, coordinates a conserved, essential calcium ion. Mutation of this glutamine to the more usual calcium ligand, glutamate, results in an increase in the K m for nitrite by around 10-fold, while V max is unaltered. Protein film voltammetry showed that lower potentials were required to detect activity from NrfA Q263E when compared with native enzyme, consistent with the introduction of a negative charge into the vicinity of the active site heme. EPR and MCD spectroscopic studies revealed the high spin state of the active site to be preserved, indicating that a water/hydroxide molecule is still coordinated to the heme in the resting state of the enzyme. Comparison of the X-ray crystal structures of the as-prepared, oxidized native and mutant enzymes showed an increased bond distance between the active site heme Fe(III) iron and the distal ligand in the latter as well as changes to the structure and mobility of the active site water molecule network. These results suggest that an important function of the unusual Q263-calcium ion pair is to increase substrate affinity through its role in supporting a network of hydrogen bonded water molecules stabilizing the active site heme distal ligand.     

Transient formation of ubisemiquinone radical and subsequent electron transfer process in the Escherichia coli cytochrome bo

To elucidate a unique mechanism for the quinol oxidation in the Escherichia coli cytochrome bo, we applied pulse radiolysis technique to the wild-type enzyme with or without a single bound ubiquinone-8 at the high-affinity quinone binding site (Q(H)), using N-methylnicotinamide (NMA) as an electron mediator. With the ubiquinone bound enzyme, the reduction of the oxidase occurred in two phases as judged from kinetic difference spectra. In the faster phase, the transient species with an absorption maximum at 440 nm, a characteristic of the formation of ubisemiquinone anion radical, appeared within 10 micros after pulse radiolysis. In the slower phase, a decrease of absorption at 440 nm was accompanied by an increase of absorption at 428 and 561 nm, characteristic of the reduced form. In contrast, with the bound ubiquinone-8-free wild-type enzyme, NMA radicals directly reduced hemes b and o, though the reduction yield was low. These results indicate that a pathway for an intramolecular electron transfer from ubisemiquinone anion radical at the Q(H) site to heme b exists in cytochrome bo. The first-order rate constant of this process was calculated to be 1.5 x 10(3) s(-1) and is comparable to a turnover rate for ubiquinol-1. The rate constant for the intramolecular electron transfer decreased considerably with increasing pH, though the yields of the formation of ubisemiquinone anion radical and the subsequent reduction of the hemes were not affected. The pH profile was tightly linked to the stability of the bound ubisemiquinone in cytochrome bo [Ingledew, W. J., Ohnishi, T., and Salerno, J. C. (1995) Eur. J. Biochem. 227, 903-908], indicating that electron transfer from the bound ubisemiquinone at the Q(H) site to the hemes slows down at the alkaline pH where the bound ubisemiquinone can be stabilized. These findings are consistent with our previous proposal that the bound ubiquinone at the Q(H) site mediates electron transfer from the low-affinity quinol oxidation site in subunit II to low-spin heme b in subunit I.     

Structure and spectroscopy of the periplasmic cytochrome c nitrite reductase from Escherichia coli

The crystal structure and spectroscopic properties of the periplasmic penta-heme cytochrome c nitrite reductase (NrfA) of Escherichia coli are presented. The structure is the first for a member of the NrfA subgroup that utilize a soluble penta-heme cytochrome, NrfB, as a redox partner. Comparison to the structures of Wolinella succinogenes NrfA and Sulfospirillum deleyianum NrfA, which accept electrons from a membrane-anchored tetra-heme cytochrome (NrfH), reveals notable differences in the protein surface around heme 2, which may be the docking site for the redox partner. The structure shows that four of the NrfA hemes (hemes 2-5) have bis-histidine axial heme-Fe ligation. The catalytic heme-Fe (heme 1) has a lysine distal ligand and an oxygen atom proximal ligand. Analysis of NrfA in solution by magnetic circular dichroism (MCD) suggested that the oxygen ligand arose from water. Electron paramagnetic resonance (EPR) spectra were collected from electrochemically poised NrfA samples. Broad perpendicular mode signals at g similar 10.8 and 3.5, characteristic of weakly spin-coupled S = 5/2, S = 1/2 paramagnets, titrated with E(m) = -107 mV. A possible origin for these are the active site Lys-OH(2) coordinated heme (heme 1) and a nearby bis-His coordinated heme (heme 3). A rhombic heme Fe(III) EPR signal at g(z) = 2.91, g(y) = 2.3, g(x) = 1.5 titrated with E(m) = -37 mV and is likely to arise from bis-His coordinated heme (heme 2) in which the interplanar angle of the imidazole rings is 21.2. The final two bis-His coordinated hemes (hemes 4 and 5) have imidazole interplanar angles of 64.4 and 71.8. Either, or both, of these hemes could give rise to a "Large g max" EPR signal at g(z)() = 3.17 that titrated at potentials between -250 and -400 mV. Previous spectroscopic studies on NrfA from a number of bacterial species are considered in the light of the structure-based spectro-potentiometric analysis presented for the E. coli NrfA.