Reaction Intermediates and Single Turnover Rate Constants for the Oxidation of Heme by Human Heme Oxygenase-1
2000; Elsevier BV; Volume: 275; Issue: 8 Linguagem: Inglês
10.1074/jbc.275.8.5297
ISSN1083-351X
AutoresYi Liu, Paul R. Ortiz de Montellano,
Tópico(s)Alcohol Consumption and Health Effects
ResumoHeme oxygenase converts heme to biliverdin, iron, and CO in a reaction with two established intermediates, α-meso-hydroxyheme and verdoheme. Transient kinetic studies show that the conversion of Fe3+-heme to Fe3+-verdoheme is biphasic. Electron transfer to the heme (0.11 s−1 at 4 °C and 0.49 s−1 at 25 °C) followed by rapid O2 binding yields the ferrous dioxy complex. Transfer of an electron (0.056 s−1 at 4 °C and 0.21 s−1 at 25 °C) to this complex triggers the formation of α-meso-hydroxyheme and its subsequent O2-dependent fragmentation to Fe3+-verdoheme. The conversion of Fe3+-verdoheme to Fe3+-biliverdin is also biphasic. Thus, reduction of Fe3+ to Fe2+-verdoheme (0.15 s−1 at 4 °C and 0.55 s−1 at 25 °C) followed by O2 binding and an electron transfer produces Fe3+-biliverdin (0.025 s−1 at 4 °C and 0.10 s−1 at 25 °C). The conversion of Fe3+-biliverdin to free biliverdin is triphasic. Reduction of Fe3+-biliverdin (0.035 s−1 at 4 °C and 0.15 s−1 at 25 °C), followed by rapid release of Fe2+ (0.19 s−1 at 4 °C and 0.39 s−1 at 25 °C), yields the biliverdin-enzyme complex from which biliverdin slowly dissociates (0.007 s−1 at 4 °C and 0.03 s−1 at 25 °C). The rate of Fe2+ release agrees with the rate of Fe3+-biliverdin reduction. Fe2+ release clearly precedes biliverdin dissociation. In the absence of biliverdin reductase, biliverdin release is the rate-limiting step, but in its presence biliverdin release is accelerated and the overall rate of heme degradation is limited by the conversion of Fe2+-verdoheme to the Fe3+-biliverdin. Heme oxygenase converts heme to biliverdin, iron, and CO in a reaction with two established intermediates, α-meso-hydroxyheme and verdoheme. Transient kinetic studies show that the conversion of Fe3+-heme to Fe3+-verdoheme is biphasic. Electron transfer to the heme (0.11 s−1 at 4 °C and 0.49 s−1 at 25 °C) followed by rapid O2 binding yields the ferrous dioxy complex. Transfer of an electron (0.056 s−1 at 4 °C and 0.21 s−1 at 25 °C) to this complex triggers the formation of α-meso-hydroxyheme and its subsequent O2-dependent fragmentation to Fe3+-verdoheme. The conversion of Fe3+-verdoheme to Fe3+-biliverdin is also biphasic. Thus, reduction of Fe3+ to Fe2+-verdoheme (0.15 s−1 at 4 °C and 0.55 s−1 at 25 °C) followed by O2 binding and an electron transfer produces Fe3+-biliverdin (0.025 s−1 at 4 °C and 0.10 s−1 at 25 °C). The conversion of Fe3+-biliverdin to free biliverdin is triphasic. Reduction of Fe3+-biliverdin (0.035 s−1 at 4 °C and 0.15 s−1 at 25 °C), followed by rapid release of Fe2+ (0.19 s−1 at 4 °C and 0.39 s−1 at 25 °C), yields the biliverdin-enzyme complex from which biliverdin slowly dissociates (0.007 s−1 at 4 °C and 0.03 s−1 at 25 °C). The rate of Fe2+ release agrees with the rate of Fe3+-biliverdin reduction. Fe2+ release clearly precedes biliverdin dissociation. In the absence of biliverdin reductase, biliverdin release is the rate-limiting step, but in its presence biliverdin release is accelerated and the overall rate of heme degradation is limited by the conversion of Fe2+-verdoheme to the Fe3+-biliverdin. iron protoporphyrin IX regardless of oxidation and ligation state heme oxygenase isoform 1 truncated human HO-1 electron paramagnetic resonance NADPH-cytochrome P450 reductase 4-morpholinepropanesulfonic acid Heme oxygenase catalyzes the NADPH and P450 reductase-dependent oxidation of heme1 to biliverdin, iron, and CO (1.Tenhunen R. Marver H.S. Schmid R. J. Biol. Chem. 1969; 244: 6388-6394Abstract Full Text PDF PubMed Google Scholar) (Scheme FS1). The enzyme, which employs heme as both the prosthetic group and substrate, regiospecifically oxidizes the heme at the α-mesoposition. This enzyme is physiologically important, in part because of the biological properties of its organic reaction products. Biliverdin is reduced by biliverdin reductase to bilirubin, which is then excreted as the glucuronic acid conjugate (2.Schmid R. McDonagh A.F. Dolphin D. The Porphyrins. 6. Academic Press, New York1979: 257-292Google Scholar). The excretion of bilirubin is frequently impaired in newborn children as well as in individuals with genetic glucuronyltransferase deficiencies (3.Maines M.D. Heme Oxygenase: Clinical Applications and Functions. CRC Press, Inc., Boca Raton, FL1992: 203-266Google Scholar). High concentrations of unconjugated bilirubin are neurotoxic, and the prevention of its accumulation through phototherapy or inhibition of heme oxygenase is of clinical importance (4.Maines M.D. Trakshel G.M. Biochim. Biophys. Acta. 1992; 1131: 166-174Crossref PubMed Scopus (61) Google Scholar, 5.Kappas A. Drummond G.S. Manola T. Petmezaki S. Values T. Pediatrics. 1988; 81: 485-497PubMed Google Scholar, 6.Drummond G.S. Kappas A. Proc. Natl. Acad. Sci. U. S. A. 1981; 78: 6466-6470Crossref PubMed Scopus (271) Google Scholar). CO, the other organic product of heme oxygenase, appears to play a role akin to nitric oxide as a signaling molecule (7.Verma A. Hirsch D.L. Glatt C.E. Ronnett G.V. Snyder S.H. Science. 1993; 259: 381-384Crossref PubMed Scopus (1364) Google Scholar, 8.Stevens C.F. Wang Y. Nature. 1993; 364: 147-149Crossref PubMed Scopus (243) Google Scholar, 9.Zhuo M. Small S.A. Kandel E.R. Hawkins R.D. Science. 1993; 260: 1946-1950Crossref PubMed Scopus (519) Google Scholar). A role for CO in signaling pathways has received strong support from recent studies with heme oxygenase knockout mice (10.Burnett A.L. Johns D.G. Kriegsfield L.J. Klein S.L. Calvin D.C. Demas G.E. Schramm L.P. Tonegawa S. Nelson R.J. Snyder S.H. Poss K.D. Nat. Med. 1998; 4: 84-87Crossref PubMed Scopus (103) Google Scholar, 11.Zakhary R. Poss K.D. Jaffrey S.R. Ferris C.D. Tonegawa S. Snyder S.H. Proc. Natl. Acad. Sci. U. S. A. 1997; 94: 14848-14853Crossref PubMed Scopus (213) Google Scholar).The existence of two heme oxygenase isoforms, HO-1 and HO-2, is well established (12.Maines M.D. Trakshel G.M. Kutty R.K. J. Biol. Chem. 1986; 261: 411-419Abstract Full Text PDF PubMed Google Scholar, 13.Maines M.D. FASEB J. 1988; 2: 2557-2568Crossref PubMed Scopus (1564) Google Scholar, 14.Maines M.D. Heme Oxygenase: Clinical Applications and Functions. CRC Press, Inc., Boca Raton, FL1992: 109-144Google Scholar), and a third isoform whose significance is unclear has been described (15.McCoubrey W.K. Huang T.J. Maines M.D. Eur. J. Biochem. 1997; 247: 725-732Crossref PubMed Scopus (734) Google Scholar). HO-1 is induced by chemical agents and a variety of stress conditions and is found in highest concentration in the spleen and liver. HO-2 is not induced by exogenous stimuli and is found in highest concentrations in the brain and testes. The heme oxygenases are membrane-bound proteins (16.Yoshida T. Biro P. Cohen T. Müller R.M. Shibahara S. Eur. J. Biochem. 1988; 171: 457-461Crossref PubMed Scopus (268) Google Scholar, 17.McCoubrey W.K. Maines M.D. Arch. Biochem. Biophys. 1993; 302: 402-408Crossref PubMed Scopus (39) Google Scholar), but water-soluble, catalytically active versions of rat and human HO-1 without the 23 carboxyl-terminal amino acid membrane anchor have been expressed inEscherichia coli (18.Wilks A. Ortiz de Montellano P.R. J. Biol. Chem. 1993; 268: 22357-22362Abstract Full Text PDF PubMed Google Scholar, 19.Wilks A. Black S.M. Miller W.L. Ortiz de Montellano P.R. Biochemistry. 1995; 34: 4421-4427Crossref PubMed Scopus (109) Google Scholar, 20.Ito-Maki M. Ishikawa K. Mansfield Matera K. Sato M. Ikeda-Saito M. Yoshida T. Arch. Biochem. Biophys. 1993; 317: 253-258Crossref Scopus (69) Google Scholar).His-25 has been identified as the proximal iron ligand in the heme·HO-1 complex by site-directed mutagenesis and resonance Raman spectroscopy (21.Sun J. Wilks A. Ortiz de Montellano P.R. Loehr T.M. Biochemistry. 1993; 32: 14151-14157Crossref PubMed Scopus (106) Google Scholar, 22.Sun J. Loehr T.M. Wilks A. Ortiz de Montellano P.R. Biochemistry. 1994; 33: 13734-13740Crossref PubMed Scopus (117) Google Scholar, 23.Wilks A. Sun J. Loehr T.M. Ortiz de Montellano P.R. J. Am. Chem. Soc. 1995; 117: 2925-2926Crossref Scopus (56) Google Scholar, 24.Takahashi S. Wang J. Rousseau D.L. Ishikawa K. Yoshida T. Host J.R. Ikeda-Saito M. J. Biol. Chem. 1994; 269: 1010-1014Abstract Full Text PDF PubMed Google Scholar, 25.Takahashi S. Wang J. Rousseau D.L. Ishikawa K. Yoshida T. Takeuchi N. Ikeda-Saito M. Biochemistry. 1994; 33: 5531-5538Crossref PubMed Scopus (94) Google Scholar, 26.Ito-Maki M. Ishikawa K. Matera K.M. Sato M. Ikeda-Saito M. Yoshida T. Arch. Biochem. Biophys. 1995; 317: 253-258Crossref PubMed Scopus (72) Google Scholar). Replacement of the proximal histidine residue by an alanine produces a catalytically inactive protein that binds heme without providing a strong axial iron ligand (22.Sun J. Loehr T.M. Wilks A. Ortiz de Montellano P.R. Biochemistry. 1994; 33: 13734-13740Crossref PubMed Scopus (117) Google Scholar). Recovery of both the iron-histidine coordination and full catalytic activity when imidazole binds to the heme·H25A mutant confirms the role of His-25 as the proximal iron ligand (23.Wilks A. Sun J. Loehr T.M. Ortiz de Montellano P.R. J. Am. Chem. Soc. 1995; 117: 2925-2926Crossref Scopus (56) Google Scholar). The assignment of His-25 as the proximal iron ligand is confirmed by the human HO-1 crystal structure (27.Schuller D. Wilks A. Ortiz de Montellano P.R. Poulos T. Nat. Struct. Biol. 1999; 6: 860-867Crossref PubMed Scopus (307) Google Scholar).The HO-1-catalyzed oxidation of heme involves sequential α-meso-hydroxylation, oxygen-dependent fragmentation of the α-meso-hydroxyheme to verdoheme, and oxidative cleavage of verdoheme to biliverdin (28.Ortiz de Montellano P.R. Acc. Chem. Res. 1998; 31: 543-549Crossref Scopus (220) Google Scholar). The formation of α-meso-hydroxyheme (29.Liu Y. Moënne-Loccoz P. Loehr T. Ortiz de Montellano P.R. J. Biol. Chem. 1997; 272: 6909-6917Abstract Full Text Full Text PDF PubMed Scopus (114) Google Scholar, 30.Yoshida T. Noguchi M. Kikuchi G. Sano S. J. Biochem. (Toyko). 1981; 90: 125-131Crossref PubMed Scopus (52) Google Scholar, 31.Yoshinaga T. Sudo Y. Sano S. Biochem. J. 1990; 270: 659-664Crossref PubMed Scopus (26) Google Scholar) and verdoheme (18.Wilks A. Ortiz de Montellano P.R. J. Biol. Chem. 1993; 268: 22357-22362Abstract Full Text PDF PubMed Google Scholar, 32.Yoshinaga T. Sudo Y. Sano S. Biochem. J. 1990; 270: 659-664Crossref PubMed Scopus (26) Google Scholar, 33.Matera K.M. Takahashi S. Fujii H. Zhou H. Ishikawa K. Yoshimura T. Rousseau D.L. Yoshida T. Ikeda-Saito M. J. Biol. Chem. 1996; 271: 6618-6624Abstract Full Text PDF PubMed Scopus (94) Google Scholar) in the enzymatic reaction have been directly demonstrated. The intermediates and reaction steps involved in the formation of α-meso-hydroxyheme and its subsequent conversion to verdoheme have been the focus of several studies (18.Wilks A. Ortiz de Montellano P.R. J. Biol. Chem. 1993; 268: 22357-22362Abstract Full Text PDF PubMed Google Scholar, 29.Liu Y. Moënne-Loccoz P. Loehr T. Ortiz de Montellano P.R. J. Biol. Chem. 1997; 272: 6909-6917Abstract Full Text Full Text PDF PubMed Scopus (114) Google Scholar, 33.Matera K.M. Takahashi S. Fujii H. Zhou H. Ishikawa K. Yoshimura T. Rousseau D.L. Yoshida T. Ikeda-Saito M. J. Biol. Chem. 1996; 271: 6618-6624Abstract Full Text PDF PubMed Scopus (94) Google Scholar, 34.Wilks A. Torpey J. Ortiz de Montellano P.R. J. Biol. Chem. 1994; 269: 29553-29556Abstract Full Text PDF PubMed Google Scholar). α-meso-Hydroxylation, the first step, proceeds via a P450 reductase-dependent reduction of the iron to the ferrous state, binding of oxygen to the reduced iron, and a second one-electron reduction of the ferrous-dioxy complex (28.Ortiz de Montellano P.R. Acc. Chem. Res. 1998; 31: 543-549Crossref Scopus (220) Google Scholar). The resulting ferric peroxide complex undergoes electrophilic addition of the distal oxygen to the porphyrin ring to yield α-meso-hydroxyheme (28.Ortiz de Montellano P.R. Acc. Chem. Res. 1998; 31: 543-549Crossref Scopus (220) Google Scholar). The α-meso-hydroxyheme exists as a resonance mixture of the Fe(III) phenolate, Fe(III) keto anion, and Fe(II) π neutral radical. Oxygen rapidly reacts with the Fe(II) π neutral radical structure, via as yet undetected intermediates, to give Fe3+-verdoheme (29.Liu Y. Moënne-Loccoz P. Loehr T. Ortiz de Montellano P.R. J. Biol. Chem. 1997; 272: 6909-6917Abstract Full Text Full Text PDF PubMed Scopus (114) Google Scholar). Even less is known about the reaction intermediates involved in the conversion of verdoheme to biliverdin. Yoshida and Kikuchi (35.Yoshida T. Kikuchi G. J. Biol. Chem. 1978; 253: 4230-4236Abstract Full Text PDF PubMed Google Scholar) reported that the reconstituted heme·HO-1 complex was oxidized to the Fe3+-biliverdin complex instead of free biliverdin when ascorbate was used as a surrogate-reducing agent. The Fe3+-biliverdin thus obtained was readily converted to free biliverdin when NADPH and P450 reductase were added (35.Yoshida T. Kikuchi G. J. Biol. Chem. 1978; 253: 4230-4236Abstract Full Text PDF PubMed Google Scholar). However, these studies did not establish that the ferric complex is a true precursor of biliverdin in the P450 reductase-dependent oxidation of heme by heme oxygenase.Through the use of stopped-flow spectroscopy, we provide here kinetic and spectroscopic evidence on the reaction steps involved in the conversion of Fe3+-verdoheme to biliverdin. Our findings indicate that Fe3+-verdoheme is first reduced to Fe2+-verdoheme, which upon binding of oxygen and transfer of a second electron is converted to the Fe3+-biliverdin complex. One-electron reduction of the Fe3+-biliverdin complex by P450 reductase is followed by release of the ferrous iron atom. In the final step of the catalytic cycle, the metal-free biliverdin dissociates from the enzyme. Determination of the rate constants for formation of verdoheme, Fe3+-biliverdin, and the subsequent steps of the reaction establish that biliverdin dissociation is the rate-limiting step in the heme degradation pathway in the absence of biliverdin reductase. In the presence of biliverdin reductase, the overall rate of heme degradation appears to be limited by the conversion of Fe2+-verdoheme to Fe3+-biliverdin.DISCUSSIONHeme oxygenase catalyzes a complicated reaction sequence that proceeds through two well defined intermediates, consumes three molecules of O2 and five electrons provided by P450 reductase, and releases three physiologically active products. The well defined intermediates are α-meso-hydroxyheme and verdoheme, and the three physiologically relevant products are iron, CO, and biliverdin. Intermediates other than these two are likely to exist, but none have been identified.The conversion of heme to α-meso-hydroxyheme requires reduction of the heme from the ferric to the ferrous state, binding of O2, uptake of a second electron to give a ferric peroxide complex, and electrophilic addition of the peroxide to the porphyrin ring. The subsequent conversion of α-meso-hydroxyheme to ferric verdoheme requires a molecule of O2 but no other cofactor (29.Liu Y. Moënne-Loccoz P. Loehr T. Ortiz de Montellano P.R. J. Biol. Chem. 1997; 272: 6909-6917Abstract Full Text Full Text PDF PubMed Scopus (114) Google Scholar). However, although not required, α-meso-hydroxyheme could possibly be reduced to the ferrous state prior to formation of verdoheme, in which case the verdoheme is obtained in the ferrous rather than ferric state (33.Matera K.M. Takahashi S. Fujii H. Zhou H. Ishikawa K. Yoshimura T. Rousseau D.L. Yoshida T. Ikeda-Saito M. J. Biol. Chem. 1996; 271: 6618-6624Abstract Full Text PDF PubMed Scopus (94) Google Scholar). Two rate constants have been determined that encompass the sequence of steps from heme to ferric verdoheme: k 1, the rate of conversion of the ferric HO-heme complex to the ferrous dioxy complex, and k 2, the rate of conversion of the ferrous dioxy HO·heme complex to the ferric verdoheme complex. The rate constant k 1 (0.11 and 0.49 s−1at 4 and 25 °C, respectively) actually describes the rate of reduction of the ferric to the ferrous heme complex because O2 binding has been reported from flash photolysis studies to be very fast (40.Migita C.T. Matera K.M. Ikeda-Saito M. Olson J.S. Fujii H. Yoshimura T. Zhou H. Yoshida T. J. Biol. Chem. 1998; 273: 945-949Abstract Full Text Full Text PDF PubMed Scopus (72) Google Scholar). The rate constant k 2(0.056 and 0.21 s−1 at 4 and 25 °C, respectively) probably corresponds to the rate of reduction of the ferrous dioxy heme complex because conversion of α-meso-hydroxyheme to verdoheme reportedly occurs within the dead time of a stopped-flow instrument (33.Matera K.M. Takahashi S. Fujii H. Zhou H. Ishikawa K. Yoshimura T. Rousseau D.L. Yoshida T. Ikeda-Saito M. J. Biol. Chem. 1996; 271: 6618-6624Abstract Full Text PDF PubMed Scopus (94) Google Scholar). The failure of any spectroscopically detectable intermediate to accumulate between the ferrous dioxy and ferric verdoheme complexes is consistent with assignment ofk 2 to reduction of the ferrous dioxy complex.Conversion of the ferric verdoheme to the ferric biliverdin complex has been dissected into two kinetic steps. In the first step, the HO·verdoheme complex is reduced from the ferric to the ferrous state at a rate given by k 3 (0.15 and 0.55 s−1 at 4 and 25 °C, respectively). In the second step, characterized by the rate constant k 4 (0.025 and 0.10 s−1 at 4 and 25 °C, respectively), the ferrous HO·verdoheme complex is converted to the ferric biliverdin complex. The rate constant k 4 thus includes an O2 binding step and whatever intermediates intervene in cleavage of the verdoheme ring system, none of which accumulate in measurable amounts.The rate constants for the final steps of the catalytic process have been teased out in greater detail. Thus, reduction of the HO·ferric biliverdin complex is given by k 5, loss of the ferrous iron to give the metal-free biliverdin complex byk 6, and dissociation of the biliverdin from HO to regenerate the free enzyme by k 7. The values for these rate constants are 0.035, 0.19, and 0.007 s−1 at 4 °C and 0.15, 0.39, and 0.03 s−1 at 25 °C. These results provide the first clear evidence that iron release precedes biliverdin release and that the iron is released in the ferrous rather than ferric state. This is evident from the rate constants for ferrous iron release (k 6 = 0.19 s−1 at 4 °C) and biliverdin release (k 7 = 0.007 s−1 at 4 °C) and the fact that the ferric biliverdin complex does not dissociate in the time frame of the present experiments. The release of iron in the ferrous state may be physiologically important. Ferrous iron is suitable for uptake into normal transport and storage sites, whereas ferric iron has a low solubility and must be reduced before it can be similarly processed (41.Richardson D.R. Ponka P. Biochim. Biophys. Acta. 1997; 1331: 1-40Crossref PubMed Scopus (589) Google Scholar). It has been reported that in HO-1 knockout mice iron accumulates in liver and kidney and is associated with increased oxidative damage at the same time that the fraction of iron in hemoglobin decreases (42.Poss K.D. Tonegawa S. Proc. Natl. Acad. Sci. U. S. A. 1997; 94: 10919-10924Crossref PubMed Scopus (856) Google Scholar). The first report of a human with a heme oxygenase deficiency suggests that a similar derangement of iron homeostasis is present (43.Yachie A. Niida Y. Wada T. Igarashi N. Kaneda H. Toma T. Ohta K. Kasahara Y. Koizumi S. J. Clin. Invest. 1999; 103: 129-135Crossref PubMed Scopus (1081) Google Scholar). It is possible that in the absence of heme oxygenase heme is degraded in inappropriate compartments or by non-physiological, peroxidative pathways that release ferric iron in a form unsuitable for efficient physiological utilization.The rate-limiting step in the single turnover of heme oxygenase is dissociation of biliverdin from the protein. The rate constantk 7 = 0.007 s−1 at 4 °C for biliverdin dissociation is one-third as large ask 4 = 0.025 s−1, the next smallest rate constant. A similar difference is observed at 25 °C. However, at least two other factors may alter the rate-limiting step. First, the rate of binding of heme to HO-1, if slow, could limit catalytic turnover. The physiological rate of heme binding is difficult to ascertain because it is difficult to define the concentration of heme and whether it is free or bound to proteins. Second, under physiological conditions, heme oxygenase turnover occurs in the presence of biliverdin reductase, and interactions with this second protein could alter the enzyme kinetics. Indeed, we show here that biliverdin reductase accelerates biliverdin release sufficiently that the rate-limiting step becomes the conversion of ferrous verdoheme to ferric biliverdin (k 4). The increase in the biliverdin dissociation rate caused by interaction of heme oxygenase with biliverdin reductase presumably involves an allosteric weakening of the binding of biliverdin to HO-1 or even a direct transfer of biliverdin from HO-1 to biliverdin reductase. The crystal structure of human HO-1 indicates that the heme is bound in a cleft with an edge exposed to the solvent (27.Schuller D. Wilks A. Ortiz de Montellano P.R. Poulos T. Nat. Struct. Biol. 1999; 6: 860-867Crossref PubMed Scopus (307) Google Scholar). The electrostatic potential of the amino acids surrounding the cleft and exposed heme edge is positive. This led to the proposal that P450 reductase, which is negatively charged (44.Wang M. Roberts D.L. Paschke R. Shea T.M. Masters B.S.S. Kim J.-J.P. Proc. Natl. Acad. Sci. U. S. A. 1997; 94: 8411-8416Crossref PubMed Scopus (660) Google Scholar), binds directly over the exposed heme edge. It is possible that biliverdin reductase binds in a similar manner and helps to extract the biliverdin product. However, efforts to demonstrate that heme oxygenase and biliverdin reductase form a stable complex have been unsuccessful,2 indicating that any such protein-protein interaction is transient.In sum, the rate constants for seven steps in the heme oxygenase catalytic sequence have been determined in single turnover studies by stopped-flow spectroscopy. The final steps have been shown to involve mandatory reduction of the heme oxygenase ferric biliverdin complex, release of ferrous iron, and release of iron-free biliverdin. The last step of this sequence is the slowest, but it is accelerated in the presence of biliverdin reductase so that reduction of verdoheme becomes the rate-limiting step. This finding suggests that biliverdin reductase exerts an allosteric or direct effect on biliverdin release via some form of protein-protein interaction. Furthermore, release of ferrous rather than ferric iron may be an important feature of heme oxygenase catalysis in terms of the iron homeostasis of the organism. Heme oxygenase catalyzes the NADPH and P450 reductase-dependent oxidation of heme1 to biliverdin, iron, and CO (1.Tenhunen R. Marver H.S. Schmid R. J. Biol. Chem. 1969; 244: 6388-6394Abstract Full Text PDF PubMed Google Scholar) (Scheme FS1). The enzyme, which employs heme as both the prosthetic group and substrate, regiospecifically oxidizes the heme at the α-mesoposition. This enzyme is physiologically important, in part because of the biological properties of its organic reaction products. Biliverdin is reduced by biliverdin reductase to bilirubin, which is then excreted as the glucuronic acid conjugate (2.Schmid R. McDonagh A.F. Dolphin D. The Porphyrins. 6. Academic Press, New York1979: 257-292Google Scholar). The excretion of bilirubin is frequently impaired in newborn children as well as in individuals with genetic glucuronyltransferase deficiencies (3.Maines M.D. Heme Oxygenase: Clinical Applications and Functions. CRC Press, Inc., Boca Raton, FL1992: 203-266Google Scholar). High concentrations of unconjugated bilirubin are neurotoxic, and the prevention of its accumulation through phototherapy or inhibition of heme oxygenase is of clinical importance (4.Maines M.D. Trakshel G.M. Biochim. Biophys. Acta. 1992; 1131: 166-174Crossref PubMed Scopus (61) Google Scholar, 5.Kappas A. Drummond G.S. Manola T. Petmezaki S. Values T. Pediatrics. 1988; 81: 485-497PubMed Google Scholar, 6.Drummond G.S. Kappas A. Proc. Natl. Acad. Sci. U. S. A. 1981; 78: 6466-6470Crossref PubMed Scopus (271) Google Scholar). CO, the other organic product of heme oxygenase, appears to play a role akin to nitric oxide as a signaling molecule (7.Verma A. Hirsch D.L. Glatt C.E. Ronnett G.V. Snyder S.H. Science. 1993; 259: 381-384Crossref PubMed Scopus (1364) Google Scholar, 8.Stevens C.F. Wang Y. Nature. 1993; 364: 147-149Crossref PubMed Scopus (243) Google Scholar, 9.Zhuo M. Small S.A. Kandel E.R. Hawkins R.D. Science. 1993; 260: 1946-1950Crossref PubMed Scopus (519) Google Scholar). A role for CO in signaling pathways has received strong support from recent studies with heme oxygenase knockout mice (10.Burnett A.L. Johns D.G. Kriegsfield L.J. Klein S.L. Calvin D.C. Demas G.E. Schramm L.P. Tonegawa S. Nelson R.J. Snyder S.H. Poss K.D. Nat. Med. 1998; 4: 84-87Crossref PubMed Scopus (103) Google Scholar, 11.Zakhary R. Poss K.D. Jaffrey S.R. Ferris C.D. Tonegawa S. Snyder S.H. Proc. Natl. Acad. Sci. U. S. A. 1997; 94: 14848-14853Crossref PubMed Scopus (213) Google Scholar). The existence of two heme oxygenase isoforms, HO-1 and HO-2, is well established (12.Maines M.D. Trakshel G.M. Kutty R.K. J. Biol. Chem. 1986; 261: 411-419Abstract Full Text PDF PubMed Google Scholar, 13.Maines M.D. FASEB J. 1988; 2: 2557-2568Crossref PubMed Scopus (1564) Google Scholar, 14.Maines M.D. Heme Oxygenase: Clinical Applications and Functions. CRC Press, Inc., Boca Raton, FL1992: 109-144Google Scholar), and a third isoform whose significance is unclear has been described (15.McCoubrey W.K. Huang T.J. Maines M.D. Eur. J. Biochem. 1997; 247: 725-732Crossref PubMed Scopus (734) Google Scholar). HO-1 is induced by chemical agents and a variety of stress conditions and is found in highest concentration in the spleen and liver. HO-2 is not induced by exogenous stimuli and is found in highest concentrations in the brain and testes. The heme oxygenases are membrane-bound proteins (16.Yoshida T. Biro P. Cohen T. Müller R.M. Shibahara S. Eur. J. Biochem. 1988; 171: 457-461Crossref PubMed Scopus (268) Google Scholar, 17.McCoubrey W.K. Maines M.D. Arch. Biochem. Biophys. 1993; 302: 402-408Crossref PubMed Scopus (39) Google Scholar), but water-soluble, catalytically active versions of rat and human HO-1 without the 23 carboxyl-terminal amino acid membrane anchor have been expressed inEscherichia coli (18.Wilks A. Ortiz de Montellano P.R. J. Biol. Chem. 1993; 268: 22357-22362Abstract Full Text PDF PubMed Google Scholar, 19.Wilks A. Black S.M. Miller W.L. Ortiz de Montellano P.R. Biochemistry. 1995; 34: 4421-4427Crossref PubMed Scopus (109) Google Scholar, 20.Ito-Maki M. Ishikawa K. Mansfield Matera K. Sato M. Ikeda-Saito M. Yoshida T. Arch. Biochem. Biophys. 1993; 317: 253-258Crossref Scopus (69) Google Scholar). His-25 has been identified as the proximal iron ligand in the heme·HO-1 complex by site-directed mutagenesis and resonance Raman spectroscopy (21.Sun J. Wilks A. Ortiz de Montellano P.R. Loehr T.M. Biochemistry. 1993; 32: 14151-14157Crossref PubMed Scopus (106) Google Scholar, 22.Sun J. Loehr T.M. Wilks A. Ortiz de Montellano P.R. Biochemistry. 1994; 33: 13734-13740Crossref PubMed Scopus (117) Google Scholar, 23.Wilks A. Sun J. Loehr T.M. Ortiz de Montellano P.R. J. Am. Chem. Soc. 1995; 117: 2925-2926Crossref Scopus (56) Google Scholar, 24.Takahashi S. Wang J. Rousseau D.L. Ishikawa K. Yoshida T. Host J.R. Ikeda-Saito M. J. Biol. Chem. 1994; 269: 1010-1014Abstract Full Text PDF PubMed Google Scholar, 25.Takahashi S. Wang J. Rousseau D.L. Ishikawa K. Yoshida T. Takeuchi N. Ikeda-Saito M. Biochemistry. 1994; 33: 5531-5538Crossref PubMed Scopus (94) Google Scholar, 26.Ito-Maki M. Ishikawa K. Matera K.M. Sato M. Ikeda-Saito M. Yoshida T. Arch. Biochem. Biophys. 1995; 317: 253-258Crossref PubMed Scopus (72) Google Scholar). Replacement of the proximal histidine residue by an alanine produces a catalytically inactive protein that binds heme without providing a strong axial iron ligand (22.Sun J. Loehr T.M. Wilks A. Ortiz de Montellano P.R. Biochemistry. 1994; 33: 13734-13740Crossref PubMed Scopus (117) Google Scholar). Recovery of both the iron-histidine coordination and full catalytic activity when imidazole binds to the heme·H25A mutant confirms the role of His-25 as the proximal iron ligand (23.Wilks A. Sun J. Loehr T.M. Ortiz de Montellano P.R. J. Am. Chem. Soc. 1995; 117: 2925-2926Crossref Scopus (56) Google Scholar). The assignment of His-25 as the proximal iron ligand is confirmed by the human HO-1 crystal structure (27.Schuller D. Wilks A. Ortiz de Montellano P.R. Poulos T. Nat. Struct. Biol. 1999; 6: 860-867Crossref PubMed Scopus (307) Google Scholar). The HO-1-catalyzed oxidation of heme involves sequential α-meso-hydroxylation, oxygen-dependent fragmentation of the α-meso-hydroxyheme to verdoheme, and oxidative cleavage of verdoheme to biliverdin (28.Ortiz de Montellano P.R. Acc. Chem. Res. 1998; 31: 543-549Crossref Scopus (220) Google Scholar). The formation of α-meso-hydroxyheme (29.Liu Y. Moënne-Loccoz P. Loehr T. Ortiz de Montellano P.R. J. Biol. Chem. 1997; 272: 6909-6917Abstract Full Text Full Text PDF PubMed Scopus (114) Google Scholar, 30.Yoshida T. Noguchi M. Kikuchi G. Sano S. J. Biochem. (Toyko). 1981; 90: 125-131Crossref PubMed Scopus (52) Google Scholar, 31.Yoshinaga T. Sudo Y. Sano S. Biochem. J. 1990; 270: 659-664Crossref PubMed Scopus (26) Google Scholar) and verdoheme (18.Wilks A. Ortiz de Montellano P.R. J. Biol. Chem. 1993; 268: 22357-22362Abstract Full Text PDF PubMed Google Scholar, 32.Yoshinaga T. Sudo Y. Sano S. Biochem. J. 1990; 270: 659-664Crossref PubMed Scopus (26) Google Scholar, 33.Matera K.M. Takahashi S. Fujii H. Zhou H. Ishikawa K. Yoshimura T. Rousseau D.L. Yoshida T. Ikeda-Saito M. J. Biol. Chem. 1996; 271: 6618-6624Abstract Full Text PDF PubMed Scopus (94) Google Scholar) in the enzymatic reaction have been directly demonstrated. The intermediates and reaction steps involved in the formation of α-meso-hydroxyheme and its subsequent conversion to verdoheme have been the focus of several studies (18.Wilks A. Ortiz de Montellano P.R. J. Biol. Chem. 1993; 268: 22357-22362Abstract Full Text PDF PubMed Google Scholar, 29.Liu Y. Moënne-Loccoz P. Loehr T. Ortiz de Montellano P.R. J. Biol. Chem. 1997; 272: 6909-6917Abstract Full Text Full Text PDF PubMed Scopus (114) Google Scholar, 33.Matera K.M. Takahashi S. Fujii H. Zhou H. Ishikawa K. Yoshimura T. Rousseau D.L. Yoshida T. Ikeda-Saito M. J. Biol. Chem. 1996; 271: 6618-6624Abstract Full Text PDF PubMed Scopus (94) Google Scholar, 34.Wilks A. Torpey J. Ortiz de Montellano P.R. J. Biol. Chem. 1994; 269: 29553-29556Abstract Full Text PDF PubMed Google Scholar). α-meso-Hydroxylation, the first step, proceeds via a P450 reductase-dependent reduction of the iron to the ferrous state, binding of oxygen to the reduced iron, and a second one-electron reduction of the ferrous-dioxy complex (28.Ortiz de Montellano P.R. Acc. Chem. Res. 1998; 31: 543-549Crossref Scopus (220) Google Scholar). The resulting ferric peroxide complex undergoes electrophilic addition of the distal oxygen to the porphyrin ring to yield α-meso-hydroxyheme (28.Ortiz de Montellano P.R. Acc. Chem. Res. 1998; 31: 543-549Crossref Scopus (220) Google Scholar). The α-meso-hydroxyheme exists as a resonance mixture of the Fe(III) phenolate, Fe(III) keto anion, and Fe(II) π neutral radical. Oxygen rapidly reacts with the Fe(II) π neutral radical structure, via as yet undetected intermediates, to give Fe3+-verdoheme (29.Liu Y. Moënne-Loccoz P. Loehr T. Ortiz de Montellano P.R. J. Biol. Chem. 1997; 272: 6909-6917Abstract Full Text Full Text PDF PubMed Scopus (114) Google Scholar). Even less is known about the reaction intermediates involved in the conversion of verdoheme to biliverdin. Yoshida and Kikuchi (35.Yoshida T. Kikuchi G. J. Biol. Chem. 1978; 253: 4230-4236Abstract Full Text PDF PubMed Google Scholar) reported that the reconstituted heme·HO-1 complex was oxidized to the Fe3+-biliverdin complex instead of free biliverdin when ascorbate was used as a surrogate-reducing agent. The Fe3+-biliverdin thus obtained was readily converted to free biliverdin when NADPH and P450 reductase were added (35.Yoshida T. Kikuchi G. J. Biol. Chem. 1978; 253: 4230-4236Abstract Full Text PDF PubMed Google Scholar). However, these studies did not establish that the ferric complex is a true precursor of biliverdin in the P450 reductase-dependent oxidation of heme by heme oxygenase. Through the use of stopped-flow spectroscopy, we provide here kinetic and spectroscopic evidence on the reaction steps involved in the conversion of Fe3+-verdoheme to biliverdin. Our findings indicate that Fe3+-verdoheme is first reduced to Fe2+-verdoheme, which upon binding of oxygen and transfer of a second electron is converted to the Fe3+-biliverdin complex. One-electron reduction of the Fe3+-biliverdin complex by P450 reductase is followed by release of the ferrous iron atom. In the final step of the catalytic cycle, the metal-free biliverdin dissociates from the enzyme. Determination of the rate constants for formation of verdoheme, Fe3+-biliverdin, and the subsequent steps of the reaction establish that biliverdin dissociation is the rate-limiting step in the heme degradation pathway in the absence of biliverdin reductase. In the presence of biliverdin reductase, the overall rate of heme degradation appears to be limited by the conversion of Fe2+-verdoheme to Fe3+-biliverdin. DISCUSSIONHeme oxygenase catalyzes a complicated reaction sequence that proceeds through two well defined intermediates, consumes three molecules of O2 and five electrons provided by P450 reductase, and releases three physiologically active products. The well defined intermediates are α-meso-hydroxyheme and verdoheme, and the three physiologically relevant products are iron, CO, and biliverdin. Intermediates other than these two are likely to exist, but none have been identified.The conversion of heme to α-meso-hydroxyheme requires reduction of the heme from the ferric to the ferrous state, binding of O2, uptake of a second electron to give a ferric peroxide complex, and electrophilic addition of the peroxide to the porphyrin ring. The subsequent conversion of α-meso-hydroxyheme to ferric verdoheme requires a molecule of O2 but no other cofactor (29.Liu Y. Moënne-Loccoz P. Loehr T. Ortiz de Montellano P.R. J. Biol. Chem. 1997; 272: 6909-6917Abstract Full Text Full Text PDF PubMed Scopus (114) Google Scholar). However, although not required, α-meso-hydroxyheme could possibly be reduced to the ferrous state prior to formation of verdoheme, in which case the verdoheme is obtained in the ferrous rather than ferric state (33.Matera K.M. Takahashi S. Fujii H. Zhou H. Ishikawa K. Yoshimura T. Rousseau D.L. Yoshida T. Ikeda-Saito M. J. Biol. Chem. 1996; 271: 6618-6624Abstract Full Text PDF PubMed Scopus (94) Google Scholar). Two rate constants have been determined that encompass the sequence of steps from heme to ferric verdoheme: k 1, the rate of conversion of the ferric HO-heme complex to the ferrous dioxy complex, and k 2, the rate of conversion of the ferrous dioxy HO·heme complex to the ferric verdoheme complex. The rate constant k 1 (0.11 and 0.49 s−1at 4 and 25 °C, respectively) actually describes the rate of reduction of the ferric to the ferrous heme complex because O2 binding has been reported from flash photolysis studies to be very fast (40.Migita C.T. Matera K.M. Ikeda-Saito M. Olson J.S. Fujii H. Yoshimura T. Zhou H. Yoshida T. J. Biol. Chem. 1998; 273: 945-949Abstract Full Text Full Text PDF PubMed Scopus (72) Google Scholar). The rate constant k 2(0.056 and 0.21 s−1 at 4 and 25 °C, respectively) probably corresponds to the rate of reduction of the ferrous dioxy heme complex because conversion of α-meso-hydroxyheme to verdoheme reportedly occurs within the dead time of a stopped-flow instrument (33.Matera K.M. Takahashi S. Fujii H. Zhou H. Ishikawa K. Yoshimura T. Rousseau D.L. Yoshida T. Ikeda-Saito M. J. Biol. Chem. 1996; 271: 6618-6624Abstract Full Text PDF PubMed Scopus (94) Google Scholar). The failure of any spectroscopically detectable intermediate to accumulate between the ferrous dioxy and ferric verdoheme complexes is consistent with assignment ofk 2 to reduction of the ferrous dioxy complex.Conversion of the ferric verdoheme to the ferric biliverdin complex has been dissected into two kinetic steps. In the first step, the HO·verdoheme complex is reduced from the ferric to the ferrous state at a rate given by k 3 (0.15 and 0.55 s−1 at 4 and 25 °C, respectively). In the second step, characterized by the rate constant k 4 (0.025 and 0.10 s−1 at 4 and 25 °C, respectively), the ferrous HO·verdoheme complex is converted to the ferric biliverdin complex. The rate constant k 4 thus includes an O2 binding step and whatever intermediates intervene in cleavage of the verdoheme ring system, none of which accumulate in measurable amounts.The rate constants for the final steps of the catalytic process have been teased out in greater detail. Thus, reduction of the HO·ferric biliverdin complex is given by k 5, loss of the ferrous iron to give the metal-free biliverdin complex byk 6, and dissociation of the biliverdin from HO to regenerate the free enzyme by k 7. The values for these rate constants are 0.035, 0.19, and 0.007 s−1 at 4 °C and 0.15, 0.39, and 0.03 s−1 at 25 °C. These results provide the first clear evidence that iron release precedes biliverdin release and that the iron is released in the ferrous rather than ferric state. This is evident from the rate constants for ferrous iron release (k 6 = 0.19 s−1 at 4 °C) and biliverdin release (k 7 = 0.007 s−1 at 4 °C) and the fact that the ferric biliverdin complex does not dissociate in the time frame of the present experiments. The release of iron in the ferrous state may be physiologically important. Ferrous iron is suitable for uptake into normal transport and storage sites, whereas ferric iron has a low solubility and must be reduced before it can be similarly processed (41.Richardson D.R. Ponka P. Biochim. Biophys. Acta. 1997; 1331: 1-40Crossref PubMed Scopus (589) Google Scholar). It has been reported that in HO-1 knockout mice iron accumulates in liver and kidney and is associated with increased oxidative damage at the same time that the fraction of iron in hemoglobin decreases (42.Poss K.D. Tonegawa S. Proc. Natl. Acad. Sci. U. S. A. 1997; 94: 10919-10924Crossref PubMed Scopus (856) Google Scholar). The first report of a human with a heme oxygenase deficiency suggests that a similar derangement of iron homeostasis is present (43.Yachie A. Niida Y. Wada T. Igarashi N. Kaneda H. Toma T. Ohta K. Kasahara Y. Koizumi S. J. Clin. Invest. 1999; 103: 129-135Crossref PubMed Scopus (1081) Google Scholar). It is possible that in the absence of heme oxygenase heme is degraded in inappropriate compartments or by non-physiological, peroxidative pathways that release ferric iron in a form unsuitable for efficient physiological utilization.The rate-limiting step in the single turnover of heme oxygenase is dissociation of biliverdin from the protein. The rate constantk 7 = 0.007 s−1 at 4 °C for biliverdin dissociation is one-third as large ask 4 = 0.025 s−1, the next smallest rate constant. A similar difference is observed at 25 °C. However, at least two other factors may alter the rate-limiting step. First, the rate of binding of heme to HO-1, if slow, could limit catalytic turnover. The physiological rate of heme binding is difficult to ascertain because it is difficult to define the concentration of heme and whether it is free or bound to proteins. Second, under physiological conditions, heme oxygenase turnover occurs in the presence of biliverdin reductase, and interactions with this second protein could alter the enzyme kinetics. Indeed, we show here that biliverdin reductase accelerates biliverdin release sufficiently that the rate-limiting step becomes the conversion of ferrous verdoheme to ferric biliverdin (k 4). The increase in the biliverdin dissociation rate caused by interaction of heme oxygenase with biliverdin reductase presumably involves an allosteric weakening of the binding of biliverdin to HO-1 or even a direct transfer of biliverdin from HO-1 to biliverdin reductase. The crystal structure of human HO-1 indicates that the heme is bound in a cleft with an edge exposed to the solvent (27.Schuller D. Wilks A. Ortiz de Montellano P.R. Poulos T. Nat. Struct. Biol. 1999; 6: 860-867Crossref PubMed Scopus (307) Google Scholar). The electrostatic potential of the amino acids surrounding the cleft and exposed heme edge is positive. This led to the proposal that P450 reductase, which is negatively charged (44.Wang M. Roberts D.L. Paschke R. Shea T.M. Masters B.S.S. Kim J.-J.P. Proc. Natl. Acad. Sci. U. S. A. 1997; 94: 8411-8416Crossref PubMed Scopus (660) Google Scholar), binds directly over the exposed heme edge. It is possible that biliverdin reductase binds in a similar manner and helps to extract the biliverdin product. However, efforts to demonstrate that heme oxygenase and biliverdin reductase form a stable complex have been unsuccessful,2 indicating that any such protein-protein interaction is transient.In sum, the rate constants for seven steps in the heme oxygenase catalytic sequence have been determined in single turnover studies by stopped-flow spectroscopy. The final steps have been shown to involve mandatory reduction of the heme oxygenase ferric biliverdin complex, release of ferrous iron, and release of iron-free biliverdin. The last step of this sequence is the slowest, but it is accelerated in the presence of biliverdin reductase so that reduction of verdoheme becomes the rate-limiting step. This finding suggests that biliverdin reductase exerts an allosteric or direct effect on biliverdin release via some form of protein-protein interaction. Furthermore, release of ferrous rather than ferric iron may be an important feature of heme oxygenase catalysis in terms of the iron homeostasis of the organism. Heme oxygenase catalyzes a complicated reaction sequence that proceeds through two well defined intermediates, consumes three molecules of O2 and five electrons provided by P450 reductase, and releases three physiologically active products. The well defined intermediates are α-meso-hydroxyheme and verdoheme, and the three physiologically relevant products are iron, CO, and biliverdin. Intermediates other than these two are likely to exist, but none have been identified. The conversion of heme to α-meso-hydroxyheme requires reduction of the heme from the ferric to the ferrous state, binding of O2, uptake of a second electron to give a ferric peroxide complex, and electrophilic addition of the peroxide to the porphyrin ring. The subsequent conversion of α-meso-hydroxyheme to ferric verdoheme requires a molecule of O2 but no other cofactor (29.Liu Y. Moënne-Loccoz P. Loehr T. Ortiz de Montellano P.R. J. Biol. Chem. 1997; 272: 6909-6917Abstract Full Text Full Text PDF PubMed Scopus (114) Google Scholar). However, although not required, α-meso-hydroxyheme could possibly be reduced to the ferrous state prior to formation of verdoheme, in which case the verdoheme is obtained in the ferrous rather than ferric state (33.Matera K.M. Takahashi S. Fujii H. Zhou H. Ishikawa K. Yoshimura T. Rousseau D.L. Yoshida T. Ikeda-Saito M. J. Biol. Chem. 1996; 271: 6618-6624Abstract Full Text PDF PubMed Scopus (94) Google Scholar). Two rate constants have been determined that encompass the sequence of steps from heme to ferric verdoheme: k 1, the rate of conversion of the ferric HO-heme complex to the ferrous dioxy complex, and k 2, the rate of conversion of the ferrous dioxy HO·heme complex to the ferric verdoheme complex. The rate constant k 1 (0.11 and 0.49 s−1at 4 and 25 °C, respectively) actually describes the rate of reduction of the ferric to the ferrous heme complex because O2 binding has been reported from flash photolysis studies to be very fast (40.Migita C.T. Matera K.M. Ikeda-Saito M. Olson J.S. Fujii H. Yoshimura T. Zhou H. Yoshida T. J. Biol. Chem. 1998; 273: 945-949Abstract Full Text Full Text PDF PubMed Scopus (72) Google Scholar). The rate constant k 2(0.056 and 0.21 s−1 at 4 and 25 °C, respectively) probably corresponds to the rate of reduction of the ferrous dioxy heme complex because conversion of α-meso-hydroxyheme to verdoheme reportedly occurs within the dead time of a stopped-flow instrument (33.Matera K.M. Takahashi S. Fujii H. Zhou H. Ishikawa K. Yoshimura T. Rousseau D.L. Yoshida T. Ikeda-Saito M. J. Biol. Chem. 1996; 271: 6618-6624Abstract Full Text PDF PubMed Scopus (94) Google Scholar). The failure of any spectroscopically detectable intermediate to accumulate between the ferrous dioxy and ferric verdoheme complexes is consistent with assignment ofk 2 to reduction of the ferrous dioxy complex. Conversion of the ferric verdoheme to the ferric biliverdin complex has been dissected into two kinetic steps. In the first step, the HO·verdoheme complex is reduced from the ferric to the ferrous state at a rate given by k 3 (0.15 and 0.55 s−1 at 4 and 25 °C, respectively). In the second step, characterized by the rate constant k 4 (0.025 and 0.10 s−1 at 4 and 25 °C, respectively), the ferrous HO·verdoheme complex is converted to the ferric biliverdin complex. The rate constant k 4 thus includes an O2 binding step and whatever intermediates intervene in cleavage of the verdoheme ring system, none of which accumulate in measurable amounts. The rate constants for the final steps of the catalytic process have been teased out in greater detail. Thus, reduction of the HO·ferric biliverdin complex is given by k 5, loss of the ferrous iron to give the metal-free biliverdin complex byk 6, and dissociation of the biliverdin from HO to regenerate the free enzyme by k 7. The values for these rate constants are 0.035, 0.19, and 0.007 s−1 at 4 °C and 0.15, 0.39, and 0.03 s−1 at 25 °C. These results provide the first clear evidence that iron release precedes biliverdin release and that the iron is released in the ferrous rather than ferric state. This is evident from the rate constants for ferrous iron release (k 6 = 0.19 s−1 at 4 °C) and biliverdin release (k 7 = 0.007 s−1 at 4 °C) and the fact that the ferric biliverdin complex does not dissociate in the time frame of the present experiments. The release of iron in the ferrous state may be physiologically important. Ferrous iron is suitable for uptake into normal transport and storage sites, whereas ferric iron has a low solubility and must be reduced before it can be similarly processed (41.Richardson D.R. Ponka P. Biochim. Biophys. Acta. 1997; 1331: 1-40Crossref PubMed Scopus (589) Google Scholar). It has been reported that in HO-1 knockout mice iron accumulates in liver and kidney and is associated with increased oxidative damage at the same time that the fraction of iron in hemoglobin decreases (42.Poss K.D. Tonegawa S. Proc. Natl. Acad. Sci. U. S. A. 1997; 94: 10919-10924Crossref PubMed Scopus (856) Google Scholar). The first report of a human with a heme oxygenase deficiency suggests that a similar derangement of iron homeostasis is present (43.Yachie A. Niida Y. Wada T. Igarashi N. Kaneda H. Toma T. Ohta K. Kasahara Y. Koizumi S. J. Clin. Invest. 1999; 103: 129-135Crossref PubMed Scopus (1081) Google Scholar). It is possible that in the absence of heme oxygenase heme is degraded in inappropriate compartments or by non-physiological, peroxidative pathways that release ferric iron in a form unsuitable for efficient physiological utilization. The rate-limiting step in the single turnover of heme oxygenase is dissociation of biliverdin from the protein. The rate constantk 7 = 0.007 s−1 at 4 °C for biliverdin dissociation is one-third as large ask 4 = 0.025 s−1, the next smallest rate constant. A similar difference is observed at 25 °C. However, at least two other factors may alter the rate-limiting step. First, the rate of binding of heme to HO-1, if slow, could limit catalytic turnover. The physiological rate of heme binding is difficult to ascertain because it is difficult to define the concentration of heme and whether it is free or bound to proteins. Second, under physiological conditions, heme oxygenase turnover occurs in the presence of biliverdin reductase, and interactions with this second protein could alter the enzyme kinetics. Indeed, we show here that biliverdin reductase accelerates biliverdin release sufficiently that the rate-limiting step becomes the conversion of ferrous verdoheme to ferric biliverdin (k 4). The increase in the biliverdin dissociation rate caused by interaction of heme oxygenase with biliverdin reductase presumably involves an allosteric weakening of the binding of biliverdin to HO-1 or even a direct transfer of biliverdin from HO-1 to biliverdin reductase. The crystal structure of human HO-1 indicates that the heme is bound in a cleft with an edge exposed to the solvent (27.Schuller D. Wilks A. Ortiz de Montellano P.R. Poulos T. Nat. Struct. Biol. 1999; 6: 860-867Crossref PubMed Scopus (307) Google Scholar). The electrostatic potential of the amino acids surrounding the cleft and exposed heme edge is positive. This led to the proposal that P450 reductase, which is negatively charged (44.Wang M. Roberts D.L. Paschke R. Shea T.M. Masters B.S.S. Kim J.-J.P. Proc. Natl. Acad. Sci. U. S. A. 1997; 94: 8411-8416Crossref PubMed Scopus (660) Google Scholar), binds directly over the exposed heme edge. It is possible that biliverdin reductase binds in a similar manner and helps to extract the biliverdin product. However, efforts to demonstrate that heme oxygenase and biliverdin reductase form a stable complex have been unsuccessful,2 indicating that any such protein-protein interaction is transient. In sum, the rate constants for seven steps in the heme oxygenase catalytic sequence have been determined in single turnover studies by stopped-flow spectroscopy. The final steps have been shown to involve mandatory reduction of the heme oxygenase ferric biliverdin complex, release of ferrous iron, and release of iron-free biliverdin. The last step of this sequence is the slowest, but it is accelerated in the presence of biliverdin reductase so that reduction of verdoheme becomes the rate-limiting step. This finding suggests that biliverdin reductase exerts an allosteric or direct effect on biliverdin release via some form of protein-protein interaction. Furthermore, release of ferrous rather than ferric iron may be an important feature of heme oxygenase catalysis in terms of the iron homeostasis of the organism. We thank Mahin Maines (University of Rochester) and Clark Lagarias (University of California, Davis) for the rat biliverdin reductase cDNA.
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