Reaction of nitric oxide with heme proteins: studies on metmyoglobin, opossum methemoglobin, and microperoxidase

Kinetic and EPR studies show that the first step in the reaction of NO with ferric myoglobin, opossum hemoglobin, and microperoxidase is the reversible formation of the H-NO complex: H + NO in equilibrium H-NO (where H = Mb+, or Hb+ OP, or MP+). The NO-combination rates are markedly affected by the presence or absence of the distal histidine. The distal histidine significantly reduces the NO-combination rates, perhaps by interaction between the distal histidine and the ferric iron. Thus the beta-chains of Hb+ OP and metmyoglobin show similar combination rates. In the absence of a distal histidine, the NO-combination rates in the alpha-chains of Hb+ OP are much faster and similar to those observed for the five-coordinate heme in microperoxidase. The loss of a water molecule from the six-coordination site is assumed to be the rate-limiting step.     

High-valent intermediates of heme proteins and model compounds

Recent computational and experimental probes of high-valent intermediates in heme proteins and model compounds reveal a rich spectrum of chemical behavior that is dependent on the nature of the proximal ligand, metal center, distal- and proximal-binding site environment, porphyrin macrocycle architecture, and consequent electronic structure. The results of such studies reveal an underlying complexity, which is simply understood once one is cognizant of the 'chameleon'-like behavior of such intermediates is determined by the high-valent intermediate environment.     

Linear free-energy relationships in binding of oxygen and carbon monoxide with heme model compounds and heme proteins

For almost a decade heme model compounds have been designed to test the influence of proximal base restraint or of distal steric hindrance upon the ligand affinity of hemoglobins. Despite the variety of molecular structures which have been successively proposed, the evaluation of the reported data is rendered difficult because of the small number of examples available within each series. In this paper we report on the kinetics of binding of oxygen and carbon monoxide with a series of nine closely related heme models. The 'basket-handle porphyrins' allow one to modify the constraints exerted upon a chelated proximal base as well as the chemical environment of the distal side of the heme. One salient feature of these models is the possibility of introducing a hydrogen-bond stabilization of the oxygen by incorporating an amide group in the vicinity of the iron centre. The structural changes among models are sufficiently 'soft' to cause an almost continuous variation of the binding constants and rate parameters. The latter are found to obey a definite linear free energy relationship which proves that the series is homogenous from a thermodynamic viewpoint. This suggests an alternative way for comparing the trends in ligand binding in different heme model families with those of heme proteins, which is developed in the discussion using literature data.     

Structure of nitric oxide hemoglobin

We have compared the structure of horse nitric oxide hemoglobin (HbNO) and methemoglobin in the oxy quaternary structure by difference Fourier analysis at 2.8 Å resolution. Both nitric oxide and oxygen assume bent co-ordination geometry and form low-spin complexes in binding to heme; on the basis of preferred ligand and heme stereochemistry, HbNO is the closest analog of HbO₂ (oxyhemoglobin) examined to date. To the resolution of the X-ray data, the stereochemistry of the heme-NO complex in hemoglobin and the corresponding free heme complex appears similar. In contrast, the ligand pockets in hemoglobin hinder binding of cyanide and carbon monoxide in their preferred linear axial co-ordination modes and force them to assume a strained off-axis binding stereochemistry. The structural similarity between HbNO and HbO₂ is reflected in their kinetic behavior, which is similar, and distinct from that of carboxyhemoglobin.     

Interaction of nitric oxide with human heme oxygenase-1

NO and CO may complement each other as signaling molecules in some physiological situations. We have examined the binding of NO to human heme oxygenase-1 (hHO-1), an enzyme that oxidizes heme to biliverdin, CO, and free iron, to determine whether inhibition of hHO-1 by NO can contribute to the signaling interplay of NO and CO. An Fe(3+)-NO hHO-1-heme complex is formed with NO or the NO donors NOC9 or 2-(N,N-diethylamino)-diazenolate-2-oxide.sodium salt. Resonance Raman spectroscopy shows that ferric hHO-1-heme forms a 6-coordinated, low spin complex with NO. The nu(N-O) vibration of this complex detected by Fourier transform IR is only 4 cm(-1) lower than that of the corresponding metmyoglobin (met-Mb) complex but is broader, suggesting a greater degree of ligand conformational freedom. The Fe(3+)-NO complex of hHO-1 is much more stable than that of met-Mb. Stopped-flow studies indicate that k(on) for formation of the hHO-1-heme Fe(3+)-NO complex is approximately 50-times faster, and k(off) 10 times slower, than for met-Mb, resulting in K(d) = 1.4 microm for NO. NO thus binds 500-fold more tightly to ferric hHO-1-heme than to met-Mb. The hHO-1 mutations E29A, G139A, D140A, S142A, G143A, G143F, and K179A/R183A do not significantly diminish the tight binding of NO, indicating that NO binding is not highly sensitive to mutations of residues that normally stabilize the distal water ligand. As expected from the K(d) value, the enzyme is reversibly inhibited upon exposure to pathologically, and possibly physiologically, relevant concentrations of NO. Inhibition of hHO-1 by NO may contribute to the pleiotropic responses to NO and CO.     

The interplay between heme iron and protein sulfhydryls in the reaction of dimeric Scapharca inaequivalvis hemoglobin with nitric oxide

The homodimeric hemoglobin from the mollusk Scapharca inaequivalvis possesses a single reactive cysteine residue per monomer, Cys92, which is located in the subunit interface in the vicinity of the heme group. The interplay between the heme iron and Cys92 towards the reaction with NO has been investigated by the combined use of electrospray mass spectrometry, FTIR and UV-Visible spectroscopy. When the ferrous liganded or unliganded protein reacts with free NO in solution Cys92 is not modified, but undergoes nitrosation when the hemoglobin is exposed to the nitric oxide releaser S-nitrosocysteine. When the ferric protein reacts with free NO under anaerobic conditions the heme iron is reduced and Cys92 is nitrosated. At variance with other hemeproteins investigated to date, in Scapharca HbI the heme-iron NO driven reduction is not accompanied by the formation of a ferric iron nitrosyl intermediate in detectable amounts. The results are consistent with the hypothesis that the nitrosating agent is the NO(+) species, which is generated during the NO driven reduction of the ferric heme iron. The possible reaction mechanism is discussed in comparison with recent findings on human hemoglobin and myoglobin.     

Thermochromism of heme adducts of Glycera hemoglobin and some other monomeric heme proteins

The thermally induced difference spectra of myoglobin (Mb) and Glycera dibranchiata hemoglobin (Hbm) derivatives and of cytochrome-c were recorded between 4 degrees and 30 degrees C in the 390-750 nm range. Thermodynamic parameters were estimated and upper and lower temperature limiting spectra were deduced for the various heme protein derivatives' equilibria. The effective iron d-electron population divides the hemes broadly into two different groups of behavior type. In the first group, Hbm(III)N3, Hbm(III), Mb(III)(H2O), and Cytc(III) show equilibria between two spin states. The weakest coupling between the heme and the globin occurs among the second group, for Hbm(II)CO and Mb(II)CO, which in the higher temperature limit undergoes averaging of the carbonyl tilt, while an axially elongated geometry is probably accessed for Hbm(II)NO and Mb(II)NO. Examples of the less common situation of increased absorption intensity and/or low-spin states at higher temperature were found in both groups. In the case of the methyl thioglycolate low-spin adducts of Hbm(III), an acid/base equilibrium involving thioglycolate deprotonation occurs. Apparent enthalpy-entropy compensation is exhibited by all these heme derivatives, and it is suggested that the delta H degrees and delta S degrees values relate to the intimacy of coupling between the heme structure and the solvent-dependent microconformation of the globin.     

Reaction of hemoglobin with HOCl: mechanism of heme destruction and free iron release

Hypochlorous acid (HOCl) is generated by myeloperoxidase using chloride and hydrogen peroxide as substrates. HOCl and its conjugate base (OCl⁻) bind to the heme moiety of hemoglobin (Hb) and generate a transient ferric species whose formation and decay kinetics indicate it can participate in protein aggregation and heme destruction along with subsequent free iron release. The oxidation of the Hb heme moiety by OCl⁻ was accompanied by marked heme destruction as judged by the decrease in and subsequent flattening of the Soret absorbance peak at 405 nm. HOCl-mediated Hb heme depletion was confirmed by HPLC analysis and in-gel heme staining. Exposure of Hb to increasing concentrations of HOCl produced a number of porphyrin degradation products resulting from oxidative cleavage of one or more of the carbon-methene bridges of the tetrapyrrole ring, as identified by their characteristic HPLC fluorescence and LC-MS. A nonreducing denaturing SDS-PAGE showed several degrees of protein aggregation. Similarly, porphyrin degradation products were identified after exposure of red blood cells to increasing concentrations of HOCl, indicating biological relevance of this finding. This work provides a direct link between Hb heme destruction and subsequent free iron accumulation, as occurs under inflammatory conditions where HOCl is formed in substantial amounts.     

Chain equivalence in reaction of nitric oxide with hemoglobin.

Mixtures of nitric oxide and hemoglobin were prepared in a rapid freeze apparatus and analyzed by EPR spectroscopy. Spectra from samples at various degrees of saturation showed that the two subunits bound NO at equal rates. Identical results were observed in 0.1 M phosphate at pH 6.5 and 0.1 M 2,2'-bis(hydroxymethyl)-2,2',2'-nitrilotriethanol, 0.1 M NaCl at pH 7.0, both in the presence and absence of inositol hexaphosphate at either buffer condition. At subsaturating levels of NO (less than 60%), or at all levels of saturation in the presence of inositol hexaphosphate, it was found that the EPR spectrum of nitrosylhemoglobin varied with the length of time before freezing. This change was characterized by the development of a hyperfine structure at g = 2.01 which appeared with a half-time of approximately 0.4 s. Maxwell and Caughey (Maxwell, J. C., and Caughey, W. S. (1976) Biochemistry 15, 388-395) have attributed this three-line EPR hyperfine structure to the formation of a pentacoordinate ferroheme-NO complex. Corresponding slow changes were observed in the visible absorption spectrum following the binding of low levels of NO to deoxyhemoglobin or inositol hexaphosphate to fully saturated nitrosylhemoglobin. Thus it appears that NO binding to the alpha and beta subunits of deoxyhemoglobin takes place at equal rates and, under conditions favoring the T quaternary state (low saturation, presence of inositol hexaphosphate), a further slow structural change takes place, resulting in the cleavage of the iron--proximal histidine bond.     

Reaction of N-hydroxyguanidine with the ferrous-oxy state of a heme peroxidase cavity mutant: A model for the reactions of nitric oxide synthase

Yeast cytochrome c peroxidase was used to construct a model for the reactions catalyzed by the second cycle of nitric oxide synthase. The R48A/W191F mutant introduced a binding site for N-hydroxyguanidine near the distal heme face and removed the redox active Trp-191 radical site. Both the R48A and R48A/W191F mutants catalyzed the H₂O₂ dependent conversion of N-hydroxyguanidine to N-nitrosoguanidine. It is proposed that these reactions proceed by direct one-electron oxidation of NHG by the Fe⁺⁴O center of either Compound I (Fe⁺⁴O, porph⁺) or Compound ES (Fe⁺⁴O, Trp⁺). R48A/W191F formed a Fe⁺²O₂ complex upon photolysis of Fe⁺²CO in the presence of O₂, and N-hydroxyguanidine was observed to react with this species to produce products, distinct from N-nitrosoguanidine, that gave a positive Griess reaction for nitrate + nitrite, a positive Berthelot reaction for urea, and no evidence for formation of NO. It is proposed that HNO and urea are produced in analogy with reactions of nitric oxide synthase in the pterin-free state.     

Kinetics of nitric oxide binding to R-state hemoglobin

Despite earlier work indicating otherwise, some recent reports have suggested that nitric oxide (NO) binds to hemoglobin cooperatively. In particular, it has been suggested that, under physiological conditions, NO binds to the high-affinity R-state hemoglobin as much as 100 times faster than to the low-affinity T-state hemoglobin. This rapid NO binding could provide a means of preserving NO bioactivity. However, using a flash-flow photolysis technique, we have determined that the rate of NO binding to normal adult R-state hemoglobin is (2.1 +/- 0.1) x 10(7) (s(-1) M(-1)), which is essentially the same as that reported for T-state NO binding. (c)2002 Elsevier Science (USA).     

Reaction of neuronal nitric oxide synthase with the nitric oxide spin-trapping agent, iron complexed with N-dithiocarboxysarcosine.

A water-soluble iron complex with N-dithiocarboxysarcosine (Fe-DTCS) has been developed as an ESR spin-trapping agent for NO and successfully applied to ESR imaging of endogenous NO production in mice. We attempted to measure NO produced by purified neuronal NO synthase (nNOS) by this method, but could not detect NO. We speculated that Fe-DTCS inhibits NOS activity. In fact, it markedly inhibited NOS activity with an IC50 value of 9.7 +/- 0.7 microM in the citrulline-formation assay. DTCS alone did not inhibit the activity. An iron complex with N-methyl-D-glucamine dithiocarbamate, a similar spin-trapping agent for NO, also inhibited the activity, with an IC50 value of 25.1 +/- 2.9 microM. Fe-DTCS suppressed cytochrome c and ferricyanide reductase activities of nNOS, and markedly increased nNOS-mediated NADPH oxidation. Concomitantly, it accelerated oxygen consumption caused by activated nNOS. These results suggest that the ESR spin-trapping agent Fe-DTCS inhibits NO synthesis by interfering with the physiological electron flow from NADPH to nNOS heme iron.     

Chain non-equivalence in nitric oxide binding to hemoglobin

The ratio of the apparent rates of ligand binding to the α and β subunits of human hemoglobin on mixing with non-saturating amounts of nitric oxide has been measured by two independent methods. Electron spin resonance measurements permit direct determination of the ratio of the amounts of the respective chains bound by NO. In stopped-flow kinetics measurements, use was made of the known difference in the kinetic constants of α and β chains in hemoglobin in the reaction with n-butyl isocyanide. Both methods concur in indicating that the apparent association rate constant of NO is greater for the α than for the β chain.     

Bacillus anthracis secretes proteins that mediate heme acquisition from hemoglobin

Acquisition of iron is necessary for the replication of nearly all bacterial pathogens; however, iron of vertebrate hosts is mostly sequestered by heme and bound to hemoglobin within red blood cells. In Bacillus anthracis, the spore-forming agent of anthrax, the mechanisms of iron scavenging from hemoglobin are unknown. We report here that B. anthracis secretes IsdX1 and IsdX2, two NEAT domain proteins, to remove heme from hemoglobin, thereby retrieving iron for bacterial growth. Unlike other Gram-positive bacteria, which rely on cell wall anchored Isd proteins for heme scavenging, B. anthracis seems to have also evolved NEAT domain proteins in the extracellular milieu and in the bacterial envelope to provide for the passage of heme.     

The reactions of copper proteins with nitric oxide.

Nitric oxide (NO) can act as a ligand for copper atoms and may also engage in redox chemistry with the metal once bound. Furthermore NO posses an unpaired electron which can couple with the unpaired electron on Cu2+. These properties have been exploited to probe the active sites of copper-containing enzymes and proteins. We review these studies. In addition to the use as a spectroscopic probe for the active site we draw attention to the rapid reactions of NO at the copper sites in Cytochrome c oxidase (CcO) and laccase. These reactions in CcO occur in the ms time range, at low NO concentrations and in the presence of oxygen and may therefore be of physiological relevance to the control of respiration. Finally we speculate on the wider role that NO may play in regulation of an important group of Type 2 copper containing enzymes.     

Reaction of cytochrome c with nitrite and nitric oxide. A model of dissimilatory nitrite reductase.

The reaction of bovine heart ferrocytochrome c with nitrite was studied under various conditions. The reaction product was ferricytochrome c at around pH 5, whereas at around pH 3 it was Compound I, characterized by twin peaks at 529 and 563 nm of equal intensity. However, ferrocytochrome c decreased obeying first-order kinetics over the pH range examined, irrespective of the presence or absence of molecular oxygen. The apparent first-order rate constant was proportional to the square of the nitrite concentration at pH 4.4 and it increased as the pH was lowered. At pH 3 the reaction was so rapid that it had to be followed by stopped-flow and rapid-scanning techniques. The apparent rate constant at this pH was found to increase linearly with the nitrite concentration. Based on these results the active species of nitrite was concluded to be dinitrogen trioxide at pH 4.4 and nitrosonium ion, no+, at pH 3. Compound II was formed by reaction of ferrocytochrome c and NO gas at acidic and alkaline pH values. The absorption peaks were at 533 and 563 nm at pH 3, and at 538 and 567 nm at pH 12.9. This compound was also formed by reducing Compound I with reductants. Compound I prepared from ferricytochrome c and NO was stable below pH 6. However, appreciable absorption peaks for ferrocytochrome c appeared between pH 8 and 10, because Compound I was dissociated into ferrocytochrome c and NO+, and because ferrocytochrome c thus formed reacted with NO very slowly in this pH region. Saccharomyces ferricytochrome c under NO gas behaved differently from mammalian cytochrome, indicating the significance of the nature of the heme environment in determing the reactivity. Only at extreme pH values was Compound II formed exclusively and persisted. A model system for dissimilatory nitrite reductase was constructed by using bovine heart cytochrome c, nitrite and NADH plus PMS at pH 3.3, and a scheme involving cyclic turnover of ferrocytochrome c, Compound I and Compound II is presented, with kinetic parameters.