Heme P460 of hydroxylamine oxidoreductase of Nitrosomonas. Reaction with CO and H2O2

Hydroxylamine oxidoreductase (HAO) of Nitrosomonas catalyzes the dehydrogenation of NH2OH and subsequent addition of oxygen to form nitrite. HAO contains c hemes and the CO-binding heme P460 in a 7:1 ratio; dehydrogenation of NH2OH involves passage of electrons to P460 and then c hemes. We now report that electrons rapidly pass from c hemes of HAO to the P460 center and then to H2O2. This conclusion is supported by (a) inhibition of c heme oxidation with CO and (b) loss of H2O2-oxidizability of ferrous c hemes following specific destruction of heme P460. Reaction of ferrous P460 with H2O2 is rate-limiting. Activation of dioxygen for N-oxidation by ferrous HAO may involve the two-electron reduction of O2 by P460. The reaction of ferrous HAO with H2O2 was studied as it may reveal aspects of the mechanism of activation of dioxygen. Reaction of ferrous heme P460 with CO is slow and with low affinity as compared with other hemoproteins. Values for reaction of CO with enzyme were: k1, 1.1 X 10(-3) M-1 s-1 and Kd, 12 microM.     

Identification of novel hemes generated by heme A synthase: evidence for two successive monooxygenase reactions

Heme A, an obligatory cofactor in eukaryotic cytochrome c oxidase, is produced from heme B (protoheme) via two enzymatic reactions catalyzed by heme O synthase and heme A synthase. Heme O synthase is responsible for the addition of a farnesyl moiety, while heme A synthase catalyzes the oxidation of a methyl substituent to an aldehyde. We have cloned the heme O synthase and heme A synthase genes from Bacillus subtilis (ctaB and ctaA) and overexpressed them in Escherichia coli to probe the oxidative mechanism of heme A synthase. Because E. coli does not naturally produce or utilize heme A, this strategy effectively decoupled heme A biosynthesis from the native electron transfer pathway and heme A transport, allowing us to observe two previously unidentified hemes. We utilized HPLC, UV/visible spectroscopy, and tandem mass spectrometry to identify these novel hemes as derivatives of heme O containing an alcohol or a carboxylate moiety at position C8 on pyrrole ring D. We interpret these derivatives to be the putative alcohol intermediate and an overoxidized byproduct of heme A synthase. Because we have shown that all hemes produced by heme A synthase require O(2) for their synthesis, we propose that heme A synthase catalyzes the oxidation of the C8 methyl to an aldehyde group via two discrete monooxygenase reactions.     

Role of calcium in metalloenzymes: effects of calcium removal on the axial ligation geometry and magnetic properties of the catalytic diheme center in MauG

MauG is a diheme enzyme possessing a five-coordinate high-spin heme with an axial His ligand and a six-coordinate low-spin heme with His-Tyr axial ligation. A Ca(2+) ion is linked to the two hemes via hydrogen bond networks, and the enzyme activity depends on its presence. Removal of Ca(2+) altered the electron paramagnetic resonance (EPR) signals of each ferric heme such that the intensity of the high-spin heme was decreased and the low-spin heme was significantly broadened. Addition of Ca(2+) back to the sample restored the original EPR signals and enzyme activity. The molecular basis for this Ca(2+)-dependent behavior was studied by magnetic resonance and M?ssbauer spectroscopy. The results show that in the Ca(2+)-depleted MauG the high-spin heme was converted to a low-spin heme and the original low-spin heme exhibited a change in the relative orientations of its two axial ligands. The properties of these two hemes are each different than those of the heme in native MauG and are now similar to each other. The EPR spectrum of Ca(2+)-free MauG appears to describe one set of low-spin ferric heme signals with a large g(max) and g anisotropy and a greatly altered spin relaxation property. Both EPR and M?ssbauer spectroscopic results show that the two hemes are present as unusual highly rhombic low-spin hemes in Ca(2+)-depleted MauG, with a smaller orientation angle between the two axial ligand planes. These findings provide insight into the correlation of enzyme activity with the orientation of axial heme ligands and describe a role for the calcium ion in maintaining this structural orientation that is required for activity.     

The low-spin heme site of cytochrome o from Escherichia coli is promiscuous with respect to heme type.

Cytochrome o of Escherichia coli is able to incorporate two different structures of heme, either heme B (protoheme) or heme O, in its low-spin heme site. In contrast, the heme of the binuclear O2 reduction site is invariably heme O. Heme O is a newly discovered heme that is related to heme A, but with the formyl group of the latter replaced by methyl. Enzyme isolated from wild type E. coli has predominantly heme B in the low-spin site, whereas enzyme isolated from various overexpressing strains contains both types of enzyme in different proportions. In some strains, 70% of the enzyme has heme O in the low-spin site. Despite this variation in the structure of one of the prosthetic groups, the enzymatic activity and polypeptide composition of the enzyme remain virtually constant. EPR and activity data both indicate that heme B and heme O occupy the same low-spin heme site in the enzyme. With heme O in this site, the alpha-absorption band is narrower and further to the blue, and the Em,7 is lower, than when there is heme B in the site. In contrast to previous proposals, we show here that the enzyme does not exhibit significant spectral interactions between the hemes. The structural heterogeneity of the low-spin heme accounts for the variation in the optical spectra and redox properties of the enzyme as isolated from different strains of E. coli.     

A novel double heme substitution produces a functional bo3 variant of the quinol oxidase aa3 of Bacillus cereus. Purification and paratial characterization

A novel bo3-type quinol oxidase was highly purified from Bacillus cereus PYM1, a spontaneous mutant unable to synthesize heme A and therefore spectroscopically detectable cytochromes aa3 and caa3. The purified enzyme contained 12.4 nmol of heme O and 11.5 nmol of heme B mg-1 protein. The enzyme was composed of two subunits with an Mr of 51,000 and 30,000, respectively. Both subunits were immunoreactive to antibodies raised against the B cereus aa3 oxidase. Moreover, amino-terminal sequence analysis of the 30-kDa subunit revealed that the first 19 residues were identical to those from the 30-kDa subunit of the B. cereus aa3 oxidase. The purified bo3 oxidase failed to oxidize ferrrocytochrome c (neither yeast nor horse) but oxidized tetrachlorohydroquinol with an apparent Km of 498 microM, a Vmax of 21 micromol of O2 min-1mg-1, and a calculated turnover of 55 s-1. The quinol oxidase activity with tetrachlorohydroquinol was inhibited by potassium cyanide and 2-n-heptyl 4-hydroxyquinoline-N-oxide with an I50 of 24 and 300 microM, respectively. Our results demonstrate that the bo3 oxidase of this mutant is not the product of a new operon but instead is a cytochrome aa3 apoprotein encoded by the qox operon of the aa3 oxidase of B. cereus wild type promiscuously assembled with hemes B and O replacing heme A, producing a novel bo3 cytochrome. This is the first reported example of an enzymatically active promiscuous oxidase resulting from the simultaneous substitution of its original hemes in the high and low spin sites.     

The role of high-potential iron protein and cytochrome c(8) as alternative electron donors to the reaction center of Chromatium vinosum

Under anaerobic conditions, intact cells of the purple sulfur bacterium Chromatium vinosum exhibit rapid photooxidation of the two low-potential hemes of the c-type cytochrome associated with the reaction center, after exposure to two short light flashes separated by a dark interval. Reduction of the photooxidized low-potential hemes is very slow under these conditions. On subsequent flashes, rapid photooxidation of a high-potential reaction center heme occurs and is followed by its rereduction on the millisecond time scale. Cells maintained under aerobic conditions exhibit the millisecond time scale reduction of the photooxidized high-potential heme after each flash. Cells grown autotrophically in the presence of Na(2)S and Na(2)S(2)O(3) appear to use the soluble [4Fe-4S]-containing protein, HiPIP, as the only direct electron donor to the reaction center heme under aerobic conditions. In contrast, cells grown in the presence of organic compounds, but in the absence of Na(2)S and Na(2)S(2)O(3), appear to use a soluble c-type cytochrome (most likely cytochrome c(8)) as the only electron donor to the reaction center heme under aerobic conditions. Cells grown autotrophically, in the presence of Na(2)S and Na(2)S(2)O(3), have a slightly higher ratio of HiPIP to cytochrome c(8) and a ratio of Rieske iron-sulfur protein to reaction center that is approximately one-half that of cells grown in the absence of Na(2)S and Na(2)S(2)O(3) but in the presence of organic compounds.     

Structure, function, and assembly of heme centers in mitochondrial respiratory complexes

The sequential flow of electrons in the respiratory chain, from a low reduction potential substrate to O(2), is mediated by protein-bound redox cofactors. In mitochondria, hemes-together with flavin, iron-sulfur, and copper cofactors-mediate this multi-electron transfer. Hemes, in three different forms, are used as a protein-bound prosthetic group in succinate dehydrogenase (complex II), in bc(1) complex (complex III) and in cytochrome c oxidase (complex IV). The exact function of heme b in complex II is still unclear, and lags behind in operational detail that is available for the hemes of complex III and IV. The two b hemes of complex III participate in the unique bifurcation of electron flow from the oxidation of ubiquinol, while heme c of the cytochrome c subunit, Cyt1, transfers these electrons to the peripheral cytochrome c. The unique heme a(3), with Cu(B), form a catalytic site in complex IV that binds and reduces molecular oxygen. In addition to providing catalytic and electron transfer operations, hemes also serve a critical role in the assembly of these respiratory complexes, which is just beginning to be understood. In the absence of heme, the assembly of complex II is impaired, especially in mammalian cells. In complex III, a covalent attachment of the heme to apo-Cyt1 is a prerequisite for the complete assembly of bc(1), whereas in complex IV, heme a is required for the proper folding of the Cox 1 subunit and subsequent assembly. In this review, we provide further details of the aforementioned processes with respect to the hemes of the mitochondrial respiratory complexes. This article is part of a Special Issue entitled: Cell Biology of Metals.     

Heme orientational disorder in human adult hemoglobin reconstituted with a ring fluorinated heme and its functional consequences

A ring fluorinated heme, 13,17-bis(2-carboxylatoethyl)-3,8-diethyl-2-fluoro-7,12,18-trimethyl-porphyrinatoiron(III), has been incorporated into human adult hemoglobin (Hb A). The heme orientational disorder in the individual subunits of the protein has been readily characterized using (19)F NMR and the O(2) binding properties of the protein have been evaluated through the oxygen equilibrium analysis. The equilibrated orientations of hemes in alpha- and beta- subunits of the reconstituted protein were found to be almost completely opposite to each other, and hence were largely different from those of the native and the previously reported reconstituted proteins [T. Jue, G.N. La Mar, Heme orientational heterogeneity in deuterohemin-reconstituted horse and human hemoglobin characterized by proton nuclear magnetic resonance spectroscopy, Biochem. Biophys. Res. Commun. 119 (1984) 640-645]. Despite the large difference in the degree of the heme orientational disorder in the subunits of the proteins, the O(2) affinity and the cooperativity of the protein reconstituted with 2-MF were similar to those of the proteins reconstituted with a series of hemes chemically modified at the heme 3- and 8-positions [K. Kawabe, K. Imaizumi, Z. Yoshida, K. Imai, I. Tyuma, Studies on reconstituted myoglobins and hemoglobins II. Role of the heme side chains in the oxygenation of hemoglobin, J. Biochem. 92 (1982) 1713-1722], whose O(2) affinity and cooperativity were higher and lower, respectively, relative to those of native protein. These results indicated that the heme orientational disorder could exert little effect, if any, on the O(2) affinity properties of Hb A. This finding provides new insights into structure-function relationship of Hb A.     

Preimplantation-embryo-specific cell cycle regulation is attributed to the low expression level of retinoblastoma protein

Biosynthesis of heme A, a prosthetic group of cytochrome oxidase (COX), involves an initial farnesylation of heme B. The heme O product formed in this reaction is modified by hydroxylation of the methyl group at carbon C-8 of the porphyrin ring. This reaction was proposed to be catalyzed by Cox15p, ferredoxin, and ferredoxin reductase. Oxidation of the alcohol to the corresponding aldehyde yields heme A. In the present study we have assayed heme A and heme O in yeast COX mutants. The steady state concentrations of the two hemes in the different strains studied indicate that hydroxylation of heme O, catalyzed by Cox15p, is regulated either by a subunit or assembly intermediate of COX. The heme profiles of the mutants also suggest positive regulation of heme B farnesylation by the hydroxylated intermediate formed at the subsequent step or by Cox15p itself.     

Glutamate 107 in subunit I of cytochrome bd from Escherichia coli is part of a transmembrane intraprotein pathway conducting protons from the cytoplasm to the heme b595/heme d active site

Cytochrome bd is a terminal quinol:O 2 oxidoreductase of the respiratory chain of Escherichia coli. The enzyme generates protonmotive force without proton pumping and contains three hemes, b 558, b 595, and d. A highly conserved glutamic acid residue of transmembrane helix III in subunit I, E107, was suggested to be part of a transmembrane pathway delivering protons from the cytoplasm to the oxygen-reducing site. When E107 is replaced with leucine, the hemes are retained but the ubiquinol-1-oxidase activity is lost. We compared wild-type and E107L mutant enzymes during single turnover using absorption and electrometric techniques with a microsecond time resolution. Both wild-type and E107L mutant cytochromes bd in the fully reduced state bind O 2 rapidly, but the formation of the oxoferryl species in the mutant is dramatically retarded as compared to the wild type. Intraprotein electron redistribution induced by the photolysis of CO bound to ferrous heme d in the one-electron-reduced wild-type enzyme is coupled to the membrane potential generation, whereas the mutant cytochrome bd shows no such potential generation. The E107L mutation also causes decrease of midpoint redox potentials of hemes b 595 and d by 25-30 mV and heme b 558 by approximately 70 mV. There are two protonatable groups redox-linked to hemes b 595 and d in the active site, one of which has been recently identified as E445, whereas the second group remains unknown. Here we propose that E107 is either the second group or a key residue of a proposed proton delivery pathway leading from the cytoplasm toward this second group.     

In vitro kinetics of reduction of cytochrome c554 isolated from the reaction center of the green phototrophic bacterium, Chloroflexus aurantiacus

The photochemical reaction center in the green bacterium Chloroflexus aurantiacus is similar to that found in purple phototrophic bacteria and interacts with a multiheme membrane-bound cytochrome. We have examined the kinetics of reduction of the pure solubilized reaction center cytochrome by laser flash photolysis of solutions containing lumiflavin or FMN. Reduction by lumiflavin semiquinone followed single exponential kinetics and the observed rate constant (kobs) was linearly dependent on protein concentration (k = 1.8 X 10(7) M-1s-1 heme-1). This result suggests either that the four hemes have similar reduction rate constants which cannot be resolved or that there are large differences in rate constant and only the most reactive heme (or hemes) was observed under these conditions. To determine the relative reactivities of the four hemes, we varied the extent of heme reduction at a single total protein concentration. As the hemes were progressively reduced by steady-state illumination prior to laser flash photolysis, kobs for the reaction with fully reduced lumiflavin decreased nonlinearly. Second-order rate constants for the four hemes were assigned by nonlinear least-squares analysis of kobs vs oxidized heme concentration data. The second-order rate constants obtained in this way for the highest and lowest potential hemes differed by a factor of about 20, which is larger than expected for c-type cytochromes based on redox potential alone (a factor of about 3 would be expected). This is interpreted as being due to differences in steric accessibility. Relative to the highest potential heme, which is as reactive as a typical c-type cytochrome, we estimated a steric effect of approximately twofold for heme 2, and steric effects of approximately fivefold for hemes 3 and 4. Using fully reduced FMN as reductant of oxidized cytochrome, ionic strength effects indicate a minus-minus interaction, with approximately a -2 charge near the site of reduction of the highest potential heme.     

Internal electron transfer between hemes and Cu(II) bound at cysteine beta93 promotes methemoglobin reduction by carbon monoxide

Previous studies showed that CO/H2O oxidation provides electrons to drive the reduction of oxidized hemoglobin (metHb). We report here that Cu(II) addition accelerates the rate of metHb beta chain reduction by CO by a factor of about 1000. A mechanism whereby electron transfer occurs via an internal pathway coupling CO/H2O oxidation to Fe(III) and Cu(II) reduction is suggested by the observation that the copper-induced rate enhancement is inhibited by blocking Cys-beta93 with N-ethylmaleimide. Furthermore, this internal electron-transfer pathway is more readily established at low Cu(II) concentrations in Hb Deer Lodge (beta2His --> Arg) and other species lacking His-beta2 than in Hb A0. This difference is consistent with preferential binding of Cu(II) in Hb A0 to a high affinity site involving His-beta2, which is ineffective in promoting electron exchange between Cu(II) and the beta heme iron. Effective electron transfer is thus affected by Hb type but is not governed by the R left arrow over right arrow T conformational equilibrium. The beta hemes in Cu(II)-metHb are reduced under CO at rates close to those observed for cytochrome c oxidase, where heme and copper are present together in the oxygen-binding site and where internal electron transfer also occurs.     

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.     

Electron/proton coupling in bacterial nitric oxide reductase during reduction of oxygen

The respiratory nitric oxide reductase (NOR) from Paracoccus denitrificans catalyzes the two-electron reduction of NO to N(2)O (2NO + 2H(+) + 2e(-) --> N(2)O + H(2)O), which is an obligatory step in the sequential reduction of nitrate to dinitrogen known as denitrification. NOR has four redox-active cofactors, namely, two low-spin hemes c and b, one high-spin heme b(3), and a non-heme iron Fe(B), and belongs to same superfamily as the oxygen-reducing heme-copper oxidases. NOR can also use oxygen as an electron acceptor; this catalytic activity was investigated in this study. We show that the product in the steady-state reduction of oxygen is water. A single turnover of the fully reduced NOR with oxygen was initiated using the flow-flash technique, and the progress of the reaction monitored by time-resolved optical absorption spectroscopy. Two major phases with time constants of 40 micros and 25 ms (pH 7.5, 1 mM O(2)) were observed. The rate constant for the faster process was dependent on the O(2) concentration and is assigned to O(2) binding to heme b(3) at a bimolecular rate constant of 2 x 10(7) M(-)(1) s(-)(1). The second phase (tau = 25 ms) involves oxidation of the low-spin hemes b and c, and is coupled to the uptake of protons from the bulk solution. The rate constant for this phase shows a pH dependence consistent with rate limitation by proton transfer from an internal group with a pK(a) = 6.6. This group is presumably an amino acid residue that is crucial for proton transfer to the catalytic site also during NO reduction.     

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.     

Composition and function of cytochrome b559 in reaction centers of photosystem II of green plants

A review of a recent study of the spectral and thermodynamic properties of cytochrome b559 as well as of the electron transfer between b559 and photosystem II reaction center cofactors in isolated D1/D2/cytochrome b559 complex RC-2 is presented. Attention is paid to the existence of intermediary-potential (IP, +150 mV) and extra-low-potential (XLP, –45 mV) hemes located close to the acceptor (quinone) and donor (P680) sides of the reaction center cofactors, respectively. These hemes found in isolated RC-2 probably correspond to the high-potential and low-potential hemes in chloroplasts, respectively. The above location of the hemes is believed to allow the photoreduction of the XLP heme and photooxidation of the IP heme. The electron transfer between the two hemes is discussed in terms of the cyclic electron flow and possible involvement in water splitting.     

Rates of energy transfer between tryptophans and hemes in hemoglobin, assuming that the heme is a planar oscillator.

Using the Förster equations we have estimated the rate of energy transfer from tryptophans to hemes in hemoglobin. Assuming an isotropic distribution of the transition moments of the heme in the plane of the porphyrin, we computed the orientation factors and the consequent transfer rates from the crystallographic coordinates of human oxy- and deoxy-hemoglobin. It appears that the orientation factors do not play a limiting role in regulating the energy transfer and that the rates are controlled almost exclusively by the intrasubunit separations between tryptophans and hemes. In intact hemoglobin tetramers the intrasubunit separations are such as to reduce lifetimes to 5 and 15 ps/ns of tryptophan lifetime. Lifetimes of several hundred picoseconds would be allowed by the intersubunit separations, but intersubunits transfer becomes important only when one heme per tetramer is absent or does not accept transfer. If more than one heme per tetramer is absent lifetimes of more than 1 ns would appear.     

Intra-subunit and inter-subunit electron transfer in neuronal nitric-oxide synthase: effect of calmodulin on heterodimer catalysis

In neuronal nitric-oxide synthase (nNOS), calmodulin (CaM) binding is thought to trigger electron transfer from the reductase domain to the heme domain, which is essential for O(2) activation and NO formation. To elucidate the electron-transfer mechanism, we characterized a series of heterodimers consisting of one full-length nNOS subunit and one oxygenase-domain subunit. The results support an inter-subunit electron-transfer mechanism for the wild type nNOS, in that electrons for catalysis transfer in a Ca(2+)/CaM-dependent way from the reductase domain of one subunit to the heme of the other subunit, as proposed for inducible NOS. This suggests that the two different isoforms form similar dimeric complexes. In a series of heterodimers containing a Ca(2+)/CaM-insensitive mutant (delta40), electrons transferred from the reductase domain to both hemes in a Ca(2+)/CaM-independent way. Thus, in the delta40 mutant electron transfer from the reductase domains to the heme domains can occur via both inter-subunit and intra-subunit mechanisms. However, NO formation activity was exclusively linked to inter-subunit electron transfer and was observed only in the presence of Ca(2+)/CaM. This suggests that the mechanism of activation of nNOS by CaM is not solely dependent on the activation of electron transfer to the nNOS hemes but may involve additional structural factors linked to the catalytic action of the heme domain.     

The hemes of Escherichia coli nitrate reductase A (NarGHI): potentiometric effects of inhibitor binding to narI.

We have potentiometrically characterized the two hemes of Escherichia coli nitrate reductase A (NarGHI) using EPR and optical spectroscopy. NarGHI contains two hemes, a low-potential heme b(L) (E(m,7) = 20 mV; g(z)() = 3.36) and a high-potential heme b(H) (E(m, 7) = 120 mV; g(z)() = 3.76). Potentiometric analyses of the g(z)() features of the heme EPR spectra indicate that the E(m,7) values of both hemes are sensitive to the menaquinol analogue 2-n-heptyl-4-hydroxyquinoline N-oxide (HOQNO). This inhibitor causes a potential-inversion of the two hemes (for heme b(L), E(m,7) = 120 mV; for heme b(H), E(m,7) = 60 mV). This effect is corroborated by optical spectroscopy of a heme b(H)-deficient mutant (NarGHI(H56R)) in which the heme b(L) undergoes a DeltaE(m,7) of 70 mV in the presence of HOQNO. Another potent inhibitor of NarGHI, stigmatellin, elicits a moderate heme b(L) DeltaE(m,7) of 30 mV, but has no detectable effect on heme b(H). No effect is elicited by either inhibitor on the line shape or the E(m,7) values of the [3Fe-4S] cluster coordinated by NarH. When NarI is expressed in the absence of NarGH [NarI(DeltaGH)], two hemes are detected in potentiometric titrations with E(m,7) values of 37 mV (heme b(L); g(z)() = 3.15) and -178 mV (heme b(H); g(z)() = 2.92), suggesting that heme b(H) may be exposed to the aqueous milieu in the absence of NarGH. The identity of these hemes was confirmed by recording EPR spectra of NarI(DeltaGH)(H56R). HOQNO binding titrations followed by fluorescence spectroscopy suggest that in both NarGHI and NarI(DeltaGH), this inhibitor binds to a single high-affinity site with a K(d) of approximately 0.2 microM. These data support a functional model for NarGHI in which a single dissociable quinol binding site is associated with heme b(L) and is located toward the periplasmic side of NarI.     

Ligand preference and orientation in b- and c-type heme-binding proteins

Hemes are often incorporated into designed proteins. The importance of the heme ligand type and its orientation is still a matter of debate. Here, heme ligands and ligand orientation were investigated using a nonredundant (87 structures) and a redundant (1503 structures) set of structures to compare and contrast design features of natural b- and c-type heme-binding proteins. Histidine is the most common ligand. Marked differences in ligation motifs between b- and c-type hemes are higher occurrence of His-Met in c-type heme binding motifs (16.4% vs. 1.4%) and higher occurrence of exchangeable, small molecules in b-type heme binding motifs (67.6% vs. 9.9%). Histidine ligands that are part of the c-type CXXCH heme-binding motif show a distinct asymmetric distribution of orientation. They tend to point between either the heme propionates or between the NA and NB heme nitrogens. Molecular mechanics calculations show that this asymmetry is due to the bonded constraints of the covalent attachment between the heme and the protein. In contrast, the orientations of b-type hemes histidine ligands are found evenly distributed with no preference. Observed histidine heme ligand orientations show no dominating influence of electrostatic interactions between the heme propionates and the ligands. Furthermore, ligands in bis-His hemes are found more frequently perpendicular rather than parallel to each other. These correlations support energetic constraints on ligands that can be used in designing proteins.