Structural biology of heme monooxygenases

BBRC Biochemical and Biophysical Research Communications 338 (2005) 337–345 www.elsevier.com/locate/ybbrc

Review

Structural biology of heme monooxygenases Thomas L. Poulos * Departments of Molecular Biology and Biochemistry, Physiology and Biophysics and Chemistry, The Center in Chemical and Structural Biology, University of California, Irvine, Irvine, CA 92697-3900, USA Received 20 July 2005 Available online 13 September 2005

Abstract Over the past few years the number of crystal structures available for heme monooxygenases has substantially increased. Those most closely related to one another are cytochrome P450, nitric oxide synthase, and heme oxygenase. The present mini-review provides a summary of some recently published work on how crystallography and solution studies have provided new insights on function and especially the oxygen activation process. It now appears that in all three monooxygenases highly ordered solvent in the active site serves as direct proton donors to the iron-linked dioxygen; a requirement for splitting the O–O bond. This is in sharp contrast to the related peroxidase family of enzymes where strategically positioned amino acid side chains serve the function of shuttling protons. The P450camoxy-complex as well as various mutants in a complex with either oxygen or carbon monoxide have enabled a fairly detailed picture to be developed on the role of specific amino acids and conformational changes in both electron transfer and oxygen activation. Ó 2005 Elsevier Inc. All rights reserved. Keywords: P450; Peroxidase; Heme oxygenase; Nitric oxide synthase; Crystallography; Oxygen activation

The question often is posed to our undergraduates in introductory biochemistry courses why Nature chose oxygen to serve as the terminal oxidant in aerobic respiration. The answer is easy. O2 is a good oxidant and ultimately turns into water, which like oxygen is a strange substance but safe. Indeed, the oxidation of nearly all biological molecules by O2 is thermodynamically favorable. This, of course, raises the second question as to why life is compatible in the presence of so much of such a good oxidant. Why is it that we donÕt spontaneously ignite? Comparing the activity of O2 with those of other diatomic gases with similar thermodynamics, the 2 electron reduction of I2 has a redox potential of 0.535 V while that for the 2 electron reduction of O2 is 0.682 V. Despite this very similar favorable 2 electron reduction, we would not survive in an atmosphere consisting of 20% I2 but we do fine in 20% O2. The answer to this thermodynamic conundrum is, of course, that O2 is a paramagnetic molecule and the reaction of O2 with nearly all biological organic molecules *

Fax: +1 949 824 3280. E-mail address: [email protected].

0006-291X/$ - see front matter Ó 2005 Elsevier Inc. All rights reserved. doi:10.1016/j.bbrc.2005.07.204

is a spin forbidden process requiring a large activation energy. Thus, the students are told, we are one spin from oblivion. Hopefully they respond with a certain sense of awe and enlightenment. This presents Nature with the challenge of how to overcome the large activation energy barrier for O2 reduction without adding heat. One answer is transition metals which are excellent agents for pairing electrons and activating O2. To protect the oxygen activation-metal machinery required for oxygen activation, a complex and rich set of enzymes has evolved which can loosely be divided into porphyrin and non-porphyrin enzymes. Non-porphyrin metallo oxygenases are the leaders with respect to historical significance since the first oxygenases to be discovered by Hayaishi [1] and Mason [2] did not contain a porphyrin. From the perspective of structure–function relationships, however, heme oxygenases have played a leading role. Part of the reason is practical. The heme group is spectroscopically rich thus enabling an array of spectroscopic probes to be used to study heme enzyme intermediates. Heme enzymes, especially peroxidases, proved relatively easy to purify in large quantities. The diversity of model heme

338

T.L. Poulos / Biochemical and Biophysical Research Communications 338 (2005) 337–345

Fig. 1. The reactions catalyzed by various heme enzymes. Por, porphyrin.

chemistry also played an important role in directing attention to heme enzymes. Among heme enzymes utilizing O2 as a substrate (Fig. 1), the one that has received the most attention is cytochrome P450. This interest drives from the critical role played by P450s in a large number of biological processes and the challenge in understanding how P450s hydroxylate unactivated carbon centers in such a large array of organic substrates. More recently, nitric oxide synthase (NOS) has been shown to catalyze a P450-like reaction although NOS oxidizes only one substrate, L-Arg. Not normally included in the list of monooxygenases is heme oxygenase (HO) perhaps because the substrate for HO also is the cofactor, heme. We now know a good deal about the structure of all three heme enzyme systems thereby placing us in a position to compare and contrast structural similarities and differences related to function. This article briefly reviews some of the new insights derived from these comparative analyses. Although not a monooxygenase, some introductory comments on peroxidases are warranted. Peroxidases are close cousins

of monooxygenases and much of the language, approaches, and insights into monooxygenase chemistry had their origins with peroxidases. Peroxidases Peroxidases form stable spectroscopically distinct intermediates (Fig. 2) which is one main reason peroxidases played a major role as ideal experimental systems in the early development of enzymology. Indeed, single turnover rapid reaction stopped flow methods were worked out by Britton Chance in the 1940s [3] with peroxidases. Even to this day the peroxidase terminology spills over into the P450 community since the active hydroxylating intermediate in P450s often is called compound I. Because the various intermediates in peroxidases are relatively long lived, it has been possible to characterize these intermediates spectroscopically and, in some cases, with crystal structures. Again these intermediates have served as benchmarks for oxygenases where similar intermediates are far more difficult to trap and characterize.

Fig. 2. Molecular models of the three monooxygenases under consideration. Aside from having heme, there is no structural homology among these three enzymes.

T.L. Poulos / Biochemical and Biophysical Research Communications 338 (2005) 337–345

P450 The P450s represent the largest group of heme oxygenases in biology. A remarkable feature of P450s that is becoming increasingly clear as ever more crystal structures are solved is that the overall P450 fold is very conservative. There are two reasons why this was somewhat unexpected. First, membrane bound microsomal P450s and their soluble prokaryotic counterparts ought to have different structures, given the very different milieu in which they function. This appears not to be the case with the exception of the Nterminal membrane anchor found in microsomal P450s. Second, P450s operate on a vast array of substrates with seemingly little in common yet P450s that operate on complex and large macrolide precursors involved in antibiotic biosynthesis look very much the same as P450s that oxidize

339

much smaller molecules like camphor (Fig. 3). Indeed, the same P450s, like the drug metabolizing P450s, oxidize a variety of different substrates. It thus appears that while the P450 structure is conservative, it also is flexible with the basic elements of secondary structure being adjustable to match the substrate. Of particular importance are the F and G helices, and the loop connecting these helices (Fig. 3). The first demonstration that these regions of the molecule are flexible and undergo an open/close motion was a comparison between the P450BM3 structure with/ without a fatty acid substrate bound [4]. Since then similar motions have been observed in other P450s with the most ˚ in P4502B4 dramatic being changes of the order of 18A [5,6] as well as the demonstration of induced fit. In the thermophilic P450, CYP119, the active site actually changes shape depending on the inhibitor bound at the active site

Fig. 3. Molecular models of P450cam and P450eryF with their respective substrates. The oxygen atoms in the substrates are depicted as the darker spheres. Note that the size, shape, and polarity of the substrates are very different, yet the overall three-dimensional fold of the P450s is conserved. Key regions that do vary the most in P450s are the F and G helices, and the loop connecting these helices.

Fig. 4. The catalytic cycle of heme oxygenases. The key step which is similar to P450s and NOS is the initial hydroxylation to give a-meso-hydroxyheme. Two electrons from NADPH via P450 reductase are required for this step. The remaining steps leading to verdoheme and biliverdin are unique to heme oxygenase.

340

T.L. Poulos / Biochemical and Biophysical Research Communications 338 (2005) 337–345

[7]. This raises the intriguing possibility that those drug metabolizing P450s that operate on a large array of substrates may be able to ‘‘shape’’ themselves around various substrates. Heme oxygenase Unlike P450s, heme oxygenases (HOs) use only one substrate, heme. In the overall catalytic reaction HOs differ only in which meso heme carbon is eliminated as CO (Fig. 4). Most HOs cleave the porphyrin ring at the a-meso carbon ultimately leading to a-bilverdin. HO1 is a microsomal enzyme anchored to the membrane via a C-terminal hydrophobic tail. Most of our own effort has centered on human HO1. Cloning and expression of a construct, residues 1–265, missing the membrane anchoring tail [8] greatly facilitated mechanistic studies [9] and lead to the first structure determination of an HO [10]. Since then several others have been solved [11–14]. Like P450s and NOS, the basic HO fold is conserved in both eukaryotes and prokaryotes. HO also uses the diflavin cytochrome P450 reductase to shuttle electrons from NADPH to the HO heme. Unlike P450s, however, the axial heme ligand is His and not Cys. Nitric oxide synthase Of the 3 enzymes under consideration, nitric oxide synthase (NOS) is by far the most complex. While mammals produce only three primary NOS isoforms, a trivially small number compared to P450s, NOS is large, multidomain, and a tightly regulated enzyme. The 3 NOS isoforms participate in distinct physiological processes. Inducible NOS (iNOS) is involved in the immune system where NO is produced as a cytotoxic agent. Endothelial NOS (eNOS) is involved in the maintenance of blood pressure while neuronal NOS (nNOS) participates in neural transmission. These three isoforms have the same domain architecture, heme-FMN/FAD. The FMN/FAD domain closely resembles in primary and tertiary structure cytochrome P450 reductase [15,16]. As in P450s, NADPH reducing equivalents are funneled to the heme via first, the FAD, and then FMN. The main difference between the NOS reductase and

P450 reductase is that NOS reductase contains additional regulatory loops [17] that control enzyme activity. The linker region connecting the heme and FMN domains binds calmodulin. In the presence of Ca2+, calmodulin binds to this linker region and activates NOS activity by enabling electrons to flow from the flavins to heme. Thus, calmodulin induces some ill-defined structural change that enables the electron transfer circuit to close and the oxidation of L-Arg to NO to proceed. Both eNOS and nNOS are constitutively expressed and controlled by calmodulin. In sharp contrast, iNOS has calmodulin bound as a permanent subunit and is regulated at the level of transcription. Ligand complexes Historically, one of the main values of enzyme crystal structures is in revealing mechanistic details. This certainly was the case with peroxidases. The first heme enzyme crystal structure, cytochrome c peroxidase, immediately suggested how peroxidases work [18,19]. As expected there is an acid–base catalytic His which aids in shifting a proton from the iron-linked peroxide O1 atom to the distal O2 atom, Fe(III)–O1–O2H2, which promotes heterolysis of the O–O bond to give Fe(IV)@O in compound I. It thus was a surprise not to find a similar acid–base catalyst in the P450cam active site capable of a similar proton shuttle mechanism [20]. Moreover, like P450 neither NOS nor HO has active site groups capable of operating directly as an acid–base catalyst. Crystal structures of ligand complexes often provide important mechanistic insights. The one critical intermediate relevant to monooxygenases that can be trapped in the crystal structure is the oxy-complex. This is not without problems since many oxy-complexes are unstable and it has become increasingly clear that X-rays generate hydrated electron that can reduce the oxy-complex to give a complex mix of breakdown products that are difficult to structurally define [21]. We have been successful in trapping P450 oxy-complexes but not in NOS and HO1 although it has been possible to trap the oxy-complex in a bacterial HO [22]. In the absence of a stable oxy-complex, NO has served as reasonably good mimic for the oxy-com-

Fig. 5. Comparison of the P450cam-oxy, HO–NO, and NOS–NO complexes. A common structural feature is that a water molecule is able to directly Hbond with the ligand. It appears that in all three enzymes water plays a critical role in delivering protons to dioxygen, a required process for proper cleavage of the O–O bond.

T.L. Poulos / Biochemical and Biophysical Research Communications 338 (2005) 337–345

plex. Fig. 5 shows oxy and NO complexes of the various monooxygenases. The one common thread that ties these structures together is that a well-ordered water molecule directly H-bonds to the distal O atom of the ligand. This water very well could be the direct proton donor to dioxygen, a required step in cleavage of the O–O bond. The following intermediates are generally considered to be steps along the way to oxygen activation. FeðIIIÞ–O–O
oxy

FeðIIIÞ–O–O2
peroxy

FeðIIIÞ–O–OH2

FeðIVÞ@O

dihydroperoxy

ferryl

FeðIIIÞ–O–OH
hydroperoxy

Elegant cryogenic reduction and annealing experiments have made it possible to spectroscopically identify some of these intermediates in P450 [23], HO [24–26], and NOS [27]. Electrons and protons are both required. In order to cleave the O–O bond to give the active Fe(IV)–O intermediate, the distal leaving O atom must be di-protonated. The crystal structures in Fig. 5 strongly support the view that water is the direct proton donor to dioxygen. Various active site groups that interact with the local water structure are also critical for proper function but do not themselves directly participate as acid–base catalytic groups. Even so, the direct involvement of water may not be universal. The P450eryF-oxy structure [28] shows that water does not directly H-bond to dioxygen but instead, a substrate OH provides the only H-bond donor to dioxygen. This could be a variation on a theme where instead of water providing the needed protons, the substrate itself provides at least one proton. What remains an interesting question is why water plays such a critical role in the HO, NOS, and P450 yet plays no direct role in catalysis with peroxidases. One possible an-

341

swer is the state of protonation of the substrate. Monooxygenases begin with a proton-free substrate, O2, while peroxidases start with H2O2. Thus, the peroxidase substrate brings into the active site the required protons needed for heterolytic cleavage of the O–O bond. Peroxidases require the catalytic distal His to shuttle a proton from the O atom linked to the iron to the leaving O atom to give Fe(III)–O–O–H2 which readily undergoes heterolysis to release H2O and leave the compound I Fe(IV)@O center. In sharp contrast, monooxygenases must bring protons in from water to get to the same Fe(III)–O–OH2 intermediate. It thus appears that the monooxygenases have bypassed the use of an active site acid–base catalyst and instead use water directly. However, since peroxidases must shuttle a proton between peroxide O atoms, a strategically positioned amino acid side chain is required with His serving this function in most peroxidases. Mutants of P450cam and the oxygen activation reaction The introduction of site-specific mutagenesis in the late 1970s revolutionized the way one studies enzyme mechanisms. It became possible to dissect the role of specific amino acids in catalysis. Essential to the success of protein engineering studies are the crystal structure and the ability to recombinantly express the enzyme of interest. Even before the protein engineering era P450cam became the paradigm for P450 structure–function studies shortly after it was first described in 1968 [29]. The cloning and recombinant expression of P450cam [30] and solution of the crystal structure [20,31] ensured that most of what is known about the O2 activation process in P450s as well as other heme monooxygenases derives from studies with P450cam. The crystal structure of P450cam implicated the conserved Thr252

Fig. 6. A comparison of the oxy-complex of wild-type P450cam and the T252A and D251N mutants [39]. In going from Fe(III) to Fe(II)-oxy the peptide of Asp251 flips, the I helix opens, the 2 ‘‘catalytic’’ waters bind, and Thr252 swings into position for H-bonding to dioxygen. The same peptide flip occurs in the mutants except in D251N the helix does not open up and the water does not bind.

342

T.L. Poulos / Biochemical and Biophysical Research Communications 338 (2005) 337–345

(Fig. 6) as part of the catalytic machinery required to activate molecular oxygen. Another important residue also found in most P450s is Asp251 (Fig. 6). Both residues were early targets for mutagenesis. Changing Thr252 to Ala [32,33] eliminates enzyme activity but the rate of NADH oxidation is nearly at wild-type levels. Reducing equivalents go into the production of peroxide and water rather than substrate oxidation. Changing Asp251 to Asn also decreases enzyme activity and uncouples electron transfer from substrate hydroxylation but with this mutant the consumption of NADH is dramatically slowed [32,34,35]. Detailed proton inventory and kinetic isotope studies showed that the second electron transfer step and associated proton transfer are slowed in the D251N mutant [36]. The X-ray structures of both the T252A [37] and D251N [36] mutants have been solved and suggested that subtle changes in solvent structure may be associated with the loss in activity. Solution of the P450cam-oxy structure [21], however, made it clear that to understand the effects of mutations on the catalytic mechanism, it is important to determine structures of intermediate states, not simply the resting ferric, Fe(III), enzyme. As shown in Fig. 6, formation of the oxy-complex leads to important changes in the I helix and neighboring solvent structure. In the resting state Fe(III) structure the side chain OH of Thr252 donates an H-bond to the peptide oxygen of Gly248 (Fig. 6). Because the normal helical H-bonding pattern is disrupted near Thr252, the I helix experiences a slight kink leaving an open groove in the helix. Also note that in the Fe(III) structure the peptide of Asp251 is nearly perpendicular to the helical axis. Upon reduction and oxygen binding, the Asp251 peptide flips, the Thr252-Gly248 H-bond is broken owing to a widening of the I-helix groove, and Thr252 moves into position to H-bond with the oxygen ligand. The opening of the I-helix enables 2 new water molecules to move into position for interaction with the oxygen ligand. These waters are generally considered to be important for the proper delivery of protons to dioxygen required for dioxygen activation [21]. The mutant-oxy-complexes provide some additional important insights into the roles of Thr252 and Asp251 in oxygen activation. The T252A-oxy complex undergoes the same change in structure as wild type P450cam (Fig. 6). The D251 peptide flips and the I helix opens thus allowing the 2 catalytic waters to bind and interact with dioxygen. Therefore, the role of Thr252 cannot be to help bind the catalytic waters. In addition, in vitro mutagenesis using unnatural amino acids where the Thr252 the side chain OH is methoxylated shows that the enzyme is still active [38]. These results indicate that Thr252 cannot operate as an H-bond donor. Based on these observations, Nagano and Poulos [39] concluded that the role of Thr252 is to serve as an H-bond acceptor of the hydroperoxy intermediate, Fe(III)–OOH. In the absence of a stabilizing H-bond by Thr252, the release of peroxide rather than addition of the second proton is favored. Therefore, the T252A mutant is uncoupled but because the proton delivery machin-

ery in the form of the catalytic waters remains intact, electron/proton transfer remains fast. The situation with the D251N mutant is quite different. In this case, the 251 peptide flips (Fig. 6) but the I helix does not open up and the Thr252-Gly248 H-bond remains in place. As a result, the catalytic waters do not bind. The absence of the catalytic waters explains why the oxidation of NADH is so slow in the D251N mutant. Without a proper proton delivery mechanism proton/electron transfer slows down. This leaves open the question of why the helix fails to undergo the same changes observed in the wild type and T252A mutant. Nagano and Poulos [39] suggest that the answer lies in the Asp251 ion pairs with Arg186 and Lys178 (Fig. 6). Flipping of the D251 peptide upon dioxygen binding places an energy strain on the I helix which is relaxed by rupturing the Thr252-Gly248 H-bond, opening the helix, and binding of the catalytic waters. Throughout the transition the ion pairs between Asp251 and Lys178/ Arg186 remain intact which strictly limits the range of motions possible for relaxing the I helix strain. In other words, since Asp251 is locked down by ion pairs, the I helix is forced to open up and allow Thr252 to swing in to H-bond with dioxygen. In sharp contrast, the Asn251 side chain in the mutant is not restrained by tight ion pairs. As a result, when the 251 peptide flips there are more degrees of freedom for relaxing the I helix strain. This enables the Thr252-Gly248 H-bond to remain in place which prevents the catalytic waters from binding. Based on this reasoning, the role of Asp251 is primarily in controlling the dioxygeninduced conformational change which enables Thr252 to be properly positioned for stabilization of the hydroperoxy intermediate. This, of course, represents only one possibility. It also has been proposed that Asp251 participates more directly as a proton donor which requires some changes in structure to properly position the Asp side for interactions with the catalytic waters [21,36]. Electron transfer Another common thread that ties P450, HO, and NOS together is the electron transfer partner used to shuttle electrons from NADPH to the heme. Like P450 HO uses the FMN/FAD P450 reductase. NOS uses a very similar diflavin reductase with the exception that the reductase is linked to the C-terminal end of the heme domain and has additional loops involved with regulation of activity [17]. A good deal is known about the kinetics and forces (polar vs. non-polar) that hold these redox complexes together but there is little known about structures. The only structure of a P450 complex that is known is that formed between the heme and FMN domains of P450BM3 [40]. Even so a majority of studies have centered on P450cam. A peculiar feature of P450cam is the strict requirement for the correct redox partner, the iron–sulfur protein putidaredoxin or Pdx [41,42]. While the source of the first electron required to give the oxy-complex is not restricted to

T.L. Poulos / Biochemical and Biophysical Research Communications 338 (2005) 337–345

Pdx, the second electron transfer step requires Pdx. One explanation for this behavior is that Pdx serves an effector role as well as a source of electrons. That is, the interaction between P450cam and Pdx leads to an unspecified structural change that triggers the second electron transfer and subsequent catalytic events. Support for such structural perturbations is evidenced by changes in various spectroscopic properties of P450cam associated with changes in the distal O2 binding pocket when Pdx binds. For example, the CO stretching mode changes in the Fe(II)–CO complex when Pdx binds [43] and there are substantial changes in the proton NMR spectrum of camphor and Thr252 in helix I [44]. It generally is thought that the Pdx binds on the proximal surface of P450cam (Fig. 7) which means that structural changes indicated by the various spectral perturbations are somehow transmitted from the proximal site of Pdx binding to the opposite distal side of the heme Fig. 8. A particular mutant of P450cam, L358P, has provided unexpectedly important insights on what may happen to P450cam when Pdx binds. Tosha et al. [44] made the following observations about this mutant: (1) the proton NMR spectrum of L358P closely resembles that of wild-

343

type P450cam complexed to Pdx; (2) camphor hydroxylation by L358P can be supported by artificial reductants; (3) L358P binds Pdx about 30-fold more tightly than wild-type and; (4) the CO stretch in L358P is similar to that of wild-type P450cam. Taken together, these results suggest that the changes induced by the L358P mutation are similar to those that result from the binding of Pdx to P450cam. The crystal structure of the L358P-CO complex has been solved [45] and reveals substantial changes compared to the wild-type CO complex. The only significant change in the wild-type enzyme when CO binds is a slight movement of the substrate, camphor. In sharp contrast, CO binding to the L358P mutant leads to changes very similar to what happens when O2 binds to wild-type P450cam. That is, the peptide of Asp251 flips, the I helix opens, and the 2 ‘‘catalytic’’ waters move into the wider I helix groove. In other words, the L358P-CO complex closely resembles the wild-typeoxy-complex. The basis for this change appears to be the bulkier mutant Pro358 side chain pushing on the proximal face of the heme which in turn leads to a slight

Fig. 7. Crystal structure of the P450BM3-FMN domain complex [40] and the hypothetical model of the P450cam–Pdx complex [47]. Note that both redox partners bind to the same surface near the proximal face of the heme near the Cys ligand. The position of Leu358 is indicated in the P450cam–Pdx complex.

Fig. 8. Stereo view of the wild-type CO complex of P450cam (white model) superimposed on the L358P–CO complex (dark model). Pro358 pushes on the proximal face of the heme leading to a small movement of the heme toward the substrate in the distal pocket. This leads to a movement of the substrate and CO ligand which we assume is responsible for changes in the I helix and distal side solvent structure resulting in a distal side structure that mimics that of the wild-type oxy-complex.

344

T.L. Poulos / Biochemical and Biophysical Research Communications 338 (2005) 337–345

rotation of the substrate and reorientation of the CO ligand toward the I helix. Tosha et al. [44] and Nagano et al. [45] suggest that Pdx binding might cause a similar ‘‘push’’ on the proximal side of the heme leading to similar structural changes. What these changes have to do with electron transfer and O2 activation is somewhat more speculative. Inter-protein electron transfer reactions are controlled by several factors including the overall thermodynamic driving force and reorganization energy. The latter is the energy required to ‘‘reorganize’’ the ligands, redox active metal, solvent, and possibly protein in preparation for electron transfer. In P450cam part of the reorganization energy may be related to the I helix changes and the entry of the new catalytic waters required for O2 activation. If so then the L358P mutant may have lowered the reorganization energy making it easier for the protein to adopt the O2-activation structure. This is why artificial reductants can replace Pdx in the L358P mutant since the effector role of Pdx no longer is required. If the L358P mutant mimics what happens when Pdx binds, then the effector role of Pdx relates to distal side structural changes required for electron transfer and O2 activation. A priori the P450cam electron transfer systems appear unnecessarily complicated. Normally specificity in interprotein electron transfer is thought to be controlled by recognition and binding, and not by a complex allosteric mechanism. If so then the P450cam system is an outlier and it is a historical accident that the more complicated P450 became the paradigm for P450 structure–function studies. On the other hand, it may be only apparent that P450cam appears more complex simply because P450cam has received so much attention thus affording the opportunity for revealing both peculiar complexities as well as answering many questions. It is difficult to imagine that P450cam is the only redox system with such a level of specificity and control. Indeed, an additional level of allosteric or effector control would ensure a very high level of specificity which could well be very important in electron transfer processes involving the formation of critical metabolic intermediates. Perhaps the P450cam redox system is not so unique and others await discovery. Conclusions Heme proteins have provided a rich resource for understanding how the interaction between a ubiquitous cofactor and protein control function. Heme enzymes have been particularly important since both spectroscopic and enzymological methods can be coupled to derive important structural and functional information on intermediates. Together with protein engineering and crystallography, the field has advanced to the stage where a good deal now is known about the O2 activation process. The various intermediates on the way to O–O bond cleavage now have been identified and the critical role that solvent plays in several seemingly unrelated heme enzymes has been dem-

onstrated. What remains elusive is the nature of the active species which oxidizes the substrate. Only in peroxidases has the Fe(IV)@O species been directly observed but not in any of the monooxygenases. Heroic efforts are still underway. Meanwhile, advances in computational methods have provided important new insights on the nature of the Fe(IV)@O intermediates and the subtle differences between heme enzymes that control reactivity (see [46] for a review). The launching of this field back in 1955 by Hayaishi and Mason continues to challenge and reward as ever more sophisticated approaches are utilized to uncover such a seemingly simple process as oxygen activation. Acknowledgments Work in the Poulos laboratory was supported by grants from the National Institutes of Health. I thank current and past members of the lab who contributed to some of the work referred to in the paper including Latesh Lad, Huiying Li, Shingo Nagano, C.S. Raman, David Schuller, and Irina Sevrioukova. I also thank Dr. Huiying Li for a careful reading of the manuscript. References [1] O. Hayaishi, M. Katagiri, S. Rothberg, Mechanism of the pyrocatechase reaction, J. Am. Chem. Soc. 77 (1955) 5450–5451. [2] H.S. Mason, W.L. Powlks, E. Peterson, Oxygen transfer and electron transport by the phenolasde complex, J. Am. Chem. Soc. 77 (1955) 2914–2915. [3] B. Chance, The kinetics of the enzyme-substrate compound of peroxidase, J. Biol. Chem. 151 (1943) 553–577. [4] H. Li, T.L. Poulos, The structure of the cytochrome P450BM-3 haem domain complexed with the fatty acid substrate, palmitoleic acid, Nat. Struct. Biol. 4 (1997) 140–146. [5] E.E. Scott, Y.A. He, M.R. Wester, M.A. White, C.C. Chin, J.R. Halpert, E.F. Johnson, C.D. Stout, An open conformation of ˚ resolution, Proc. Natl. mammalian cytochrome P450 2B4 at 1.6-A Acad. Sci. USA 100 (2003) 13196–13201. [6] E.E. Scott, M.A. White, Y.A. He, E.F. Johnson, C.D. Stout, J.R. Halpert, Structure of mammalian cytochrome P450 2B4 complexed ˚ resolution: insight into the with 4-(4-chlorophenyl)imidazole at 1.9-A range of P450 conformations and the coordination of redox partner binding, J. Biol. Chem. 279 (2004) 27294–27301. [7] J. Yano, L. Koo, D. Schuller, H. Li, P.R. Ortiz de Montellano, T.L. Poulos, Crystal structure of a thermophilic cytochrome P450 from the archaeon Sulfolobus solfataricus, J. Biol. Chem. 275 (2000) 31086– 31092. [8] A. Wilks, S.M. Black, W.L. Miller, P.R. Ortiz de Montellano, Expression and characterization of truncated human heme oxygenase hHO-1 and a fusion protein of hHO-1 with human cytochrome P450 reductase, Biochemistry 34 (1995) 4421–4427. [9] P.R. Ortiz de Montellano, Heme oxygenase mechanism: evidence for an electrophilic ferric peroxide species, Acc. Chem. Res. 31 (1998) 543–549. [10] D.J. Schuller, A. Wilks, P.R. Ortiz de Montellano, T.L. Poulos, Crystal structure of human heme oxygenase-1, Nat. Struct. Biol. 6 (1999) 860–867. [11] J. Friedman, L. Lad, H. Li, A. Wilks, T.L. Poulos, Structural basis for novel delta-regioselective heme oxygenation in the opportunistic pathogen Pseudomonas aeruginosa, Biochemistry 43 (2004) 5239–5245.

T.L. Poulos / Biochemical and Biophysical Research Communications 338 (2005) 337–345 [12] M. Sugishima, H. Sakamoto, Y. Kakuta, Y. Omata, S. Hayashi, M. Noguchi, K. Fukuyama, Crystal structure of rat apo-heme oxygenase-1 (HO-1): mechanism of heme binding in HO-1 inferred from structural comparison of the apo and heme complex forms, Biochemistry 41 (2002) 7293–7300. [13] S. Hirotsu, G.C. Chu, M. Unno, D.S. Lee, T. Yoshida, S.-Y. Park, Y. Shiro, M. Ikeda-Saito, The crystal structures of the ferric and ferrous forms of the heme complex of HmuO, a heme oxygenase of Corynebacterium diphtheriae, J. Biol. Chem. 279 (2003) 11937–11947. [14] M. Sugishima, Y. Hagiwara, X. Zhang, T. Yoshida, C.T. Migita, K. Fukuyama, Crystal structure of dimeric heme oxygenase-2 from Synechocystis sp. PCC 6803 in complex with heme, Biochemistry 44 (2005) 4257–4266. [15] M. Wang, D.L. Roberts, R. Paschke, T.M. Shea, B.S. Masters, J.J. Kim, Three-dimensional structure of NADPH–cytochrome P450 reductase: prototype for FMN- and FAD-containing enzymes, Proc. Natl. Acad. Sci. USA 94 (1997) 8411–8416. [16] E.D. Garcin, C.M. Bruns, S.J. Lloyd, D.J. Hosfield, M. Tiso, R. Gachhui, D.J. Stuehr, J.A. Tainer, E.D. Getzoff, Structural basis for isozyme-specific regulation of electron transfer in nitric-oxide synthase, J. Biol. Chem. 279 (2004) 37918–37927. [17] J.C. Salerno, D.E. Harris, K. Irizarry, B. Patel, A.J. Morales, S.M. Smith, P. Martasek, L.J. Roman, B.S. Masters, C.L. Jones, et al., An autoinhibitory control element defines calcium-regulated isoforms of nitric oxide synthase, J. Biol. Chem. 272 (1997) 29769–29777. [18] T.L. Poulos, S.T. Freer, R.A. Alden, S.L. Edwards, U. Skogland, K. Takio, B. Eriksson, N. Xuong, T. Yonetani, J. Kraut, The crystal structure of cytochrome c peroxidase, J. Biol. Chem. 255 (1980) 575–580. [19] T.L. Poulos, J. Kraut, The stereochemistry of peroxidase catalysis, J. Biol. Chem. 255 (1980) 8199–8205. [20] T.L. Poulos, B.C. Finzel, I.C. Gunsalus, G.C. Wagner, J. Kraut, The ˚ crystal structure of Pseudomonas putida cytochrome P-450, J. 2.6-A Biol. Chem. 260 (1985) 16122–16130. [21] I. Schlichting, J. Berendzen, K. Chu, A.M. Stock, S.A. Maves, D.E. Benson, R.M. Sweet, D. Ringe, G.A. Petsko, S.G. Sligar, The catalytic pathway of cytochrome P450cam at atomic resolution, Science 287 (2000) 1615–1622. [22] M. Unno, T. Matsui, G.C. Chu, M. Couture, T. Yoshida, D.L. Rousseau, J.S. Olson, M. Ikeda-Saito, Crystal structure of the dioxygen-bound heme oxygenase from Corynebacterium diphtheriae: implications for heme oxygenase function, J. Biol. Chem. 279 (2004) 21055–21061. [23] R. Davydov, T.M. Makris, V. Kofman, D.E. Werst, S.G. Sligar, B.M. Hoffman, Hydroxylation of camphor by reduced oxy-cytochrome P450cam: mechanistic implications of EPR and ENDOR studies of catalytic intermediates in native and mutant enzymes, J. Am. Chem. Soc. 123 (2001) 1403–1415. [24] R.M. Davydov, T. Yoshida, M. Ikeda-Saito, B.M. Hoffman, Hydroperoxy-heme oxygenase generated by cryoreduction catalyzes the formation of a-meso-hydroxyheme as detected by EPR and ENDOR, J. Am. Chem. Soc. 121 (1999) 10656–10657. [25] R. Davydov, V. Kofman, H. Fujii, T. Yoshida, M. Ikeda-Saito, B.M. Hoffman, Catalytic mechanism of heme oxygenase through EPR and ENDOR of cryoreduced oxy-heme oxygenase and its Asp 140 mutants, J. Am. Chem. Soc 124 (2002) 1798–1808. [26] R. Davydov, T. Matsui, H. Fujii, M. Ikeda-Saito, B.M. Hoffman, Kinetic isotope effects on the rate-limiting step of heme oxygenase catalysis indicate concerted proton transfer/heme hydroxylation, J. Am. Chem. Soc. 125 (2003) 16208–16209. [27] R. Davydov, A. Ledbetter-Rogers, P. Martasek, M. Larukhin, M. Sono, J.H. Dawson, B.S. Masters, B.M. Hoffman, EPR and ENDOR characterization of intermediates in the cryoreduced oxy-nitric oxide synthase heme domain with bound L-arginine or N(G)-hydroxyarginine, Biochemistry 41 (2002) 10375–10381. [28] S. Nagano, J.R. Cupp-Vickery, T.L. Poulos, Crystal structures of the ferrous dioxygen complex of wild-type cytochrome P450eryF and its

[29]

[30]

[31] [32]

[33]

[34]

[35] [36]

[37]

[38]

[39]

[40]

[41]

[42]

[43]

[44]

[45]

[46]

[47]

345

mutants, A245S and A245T: investigation of the proton transfer system in P450eryF, J. Biol. Chem. 280 (2005) 22102–22107. M. Katagiri, B.N. Ganguli, I.C. Gunsalus, A soluble cytochrome P450 functional in methylene hydroxylation, J. Biol. Chem. 243 (1968) 3543–3546. H. Koga, B. Rauchfuss, I.C. Gunsalus, P450cam gene cloning and expression in Pseudomonas putida and Escherichia coli, Biochem. Biophys. Res. Commun. 130 (1985) 412–417. T.L. Poulos, B.C. Finzel, A.J. Howard, High-resolution crystal structure of cytochrome P450cam, J. Mol. Biol. 195 (1987) 687–700. H. Shimada, M. Imai, T. Horiuchi, Y. Ishimura, Mechanism of oxygen activation by cytochrome P-450cam, in: S.N.M. Yamamoto, M. Nozaki, Y. Ishimura (Eds.), International Symposium on Oxygenases and Oxygen Activation, Yamada Science Foundation, Osaka, Japan, 1990, pp. 133–136. S.A. Martinis, W.M. Atkins, P.S. Stayton, T.L. Poulos, A conserved residue of cytochrome P450cam is involved in heme-oxygen stability and activation, J. Am. Chem. Soc. 20 (1989) 9252–9253. N.S. Gerber, S.G. Sligar, Catalytic mechanism of cytochrome P450— evidence for a distal charge relay system, J. Am. Chem. Soc. 174 (1992) 725–735. N.C. Gerber, S.G. Sligar, A role for Asp-251 in cytochrome P-450cam oxygen activation, J. Biol. Chem. 269 (1994) 4260–4266. M. Vidakovic, S.G. Sligar, H. Li, T.L. Poulos, Understanding the role of the essential Asp251 in cytochrome p450cam using site-directed mutagenesis, crystallography, and kinetic solvent isotope effect, Biochemistry 37 (1998) 9211–9219. R. Raag, S.A. Martinis, S.G. Sligar, T.L. Poulos, Crystal structure of the cytochrome P-450CAM active site mutant Thr252Ala, Biochemistry 30 (1991) 11420–11429. Y. Kimata, H. Shimada, T. Hirose, Y. Ishimura, Role of Thr-252 in cytochrome P450cam: a study with unnatural amino acid mutagenesis, Biochem. Biophys. Res. Commun. 208 (1995) 96–102. S. Nagano, T.L. Poulos, Crystallographic study on the dioxygen complex of wild-type and mutant cytochrome P450CAM: implications for the dioxygen activation mechanism, J. Biol. Chem. 280 (2005) 31659–31663. I.F. Sevrioukova, H. Li, H. Zhang, J.A. Peterson, T.L. Poulos, Structure of a cytochrome P450-redox partner electron transfer complex, Proc. Natl. Acad. Sci. USA 96 (1999) 1863–1868. C.A. Tyson, J.D. Lipscomb, I.C. Gunsalus, The role of putidaredoxin and P450cam in methylene hydroxylation, J. Biol. Chem. 247 (1972) 5777–5784. J.D. Lipscomb, S.G. Sligar, M.J.a. Namtvedt, I.C. Gunsalus, Autooxidation and hydroxylation reactions of oxygenated cytochrome P-450cam, J. Biol. Chem. 251 (1976) 111–1124. S. Nagano, H. Shimada, A. Tarumi, T. Hishiki, Y. KimataAriga, T. Egawa, M. Suematsu, S.Y. Park, S. Adachi, Y. Shiro, Y. Ishimura, Infrared spectroscopic and mutational studies on putidaredoxin-induced conformational changes in ferrous COP450cam, Biochemistry 42 (2003) 14507–14514. T. Tosha, S. Yoshioka, K. Ishimori, I. Morishima, L358P mutation on cytochrome P450cam simulates structural changes upon Putidaredoxin binding: the structural changes trigger electron transfer to oxyP450CAMfromelectrondonors,J.Biol.Chem.279(2004)42836–42843. S. Nagano, T. Tosha, K. Ishimori, I. Morishima, T.L. Poulos, Crystal structure of the cytochrome P450cam mutant that exhibits the same spectral perturbations induced by putidaredoxin binding, J. Biol. Chem. 279 (2004) 42844–42849. S. Shaik, D. Kumar, S.P. de Visser, A. Altun, W. Thiel, Theoretical perspective on the structure and mechanism of cytochrome P450 enzymes, Chem. Rev. 105 (2005) 2279– 2328. T.C. Pochapsky, T.A. Lyons, S. Kazanis, T. Arakaki, G. Ratnaswamy, A structure-based model for cytochrome P450cam-putidaredoxin interactions, Biochimie 78 (1996) 723–733.