Regiospecificity determinants of human heme oxygenase: differential NADPH- and ascorbate-dependent heme cleavage by the R183E mutant

The ability of the human heme oxygenase-1 (hHO-1) R183E mutant to oxidize heme in reactions supported by either NADPH-cytochrome P450 reductase or ascorbic acid has been compared. The NADPH-dependent reaction, like that of wild-type hHO-1, yields exclusively biliverdin IXalpha. In contrast, the R183E mutant with ascorbic acid as the reductant produces biliverdin IXalpha (79 +/- 4%), IXdelta (19 +/- 3%), and a trace of IXbeta. In the presence of superoxide dismutase and catalase, the yield of biliverdin IXdelta is decreased to 8 +/- 1% with a corresponding increase in biliverdin IXalpha. Spectroscopic analysis of the NADPH-dependent reaction shows that the R183E ferric biliverdin complex accumulates, because reduction of the iron, which is required for sequential iron and biliverdin release, is impaired. Reversal of the charge at position 183 makes reduction of the iron more difficult. The crystal structure of the R183E mutant, determined in the ferric and ferrous-NO bound forms, shows that the heme primarily adopts the same orientation as in wild-type hHO-1. The structure of the Fe(II).NO complex suggests that an altered active site hydrogen bonding network supports catalysis in the R183E mutant. Furthermore, Arg-183 contributes to the regiospecificity of the wild-type enzyme, but its contribution is not critical. The results indicate that the ascorbate-dependent reaction is subject to a lower degree of regiochemical control than the NADPH-dependent reaction. Ascorbate may be able to reduce the R183E ferric and ferrous dioxygen complexes in active site conformations that cannot be reduced by NADPH-cytochrome P450 reductase.     

Alteration of the regiospecificity of human heme oxygenase-1 by unseating of the heme but not disruption of the distal hydrogen bonding network

Heme oxygenase regiospecifically oxidizes heme at the alpha-meso position to give biliverdin IXalpha, CO, and iron. The heme orientation within the active site, which is thought to determine the oxidation regiospecificity, is shown here for the human enzyme (hHO1) to be largely determined by interactions between the heme carboxylic acid groups and residues Arg183 and Lys18 but not Tyr134. Mutation of either Arg183 or Lys18 individually does not significantly alter the NADPH-cytochrome P450 reductase-dependent reaction regiochemistry but partially shifts the oxidation to the beta/delta-meso positions in the reaction supported by ascorbic acid. Mutation of Glu29 to a lysine, which places a positive charge where it can interact with a heme carboxyl if the heme rotates by approximately 90 degrees, causes a slight loss of regiospecificity but combined with the R183E and K18E mutations results primarily in beta/delta-meso oxidation of the heme under all conditions. NMR analysis of heme binding to the triple K18E/E29K/R183E mutant confirms rotation of the heme in the active site. Kinetic studies demonstrate that mutations of Arg183 greatly impair the rate of the P450 reductase-dependent reaction, in accord with the earlier finding that Arg183 is involved in binding of the reductase to hHO1, but have little effect on the ascorbate reaction. Mutations of Asp140 and Tyr58 that disrupt the active site hydrogen bonding network impair catalytic rates but do not influence the oxidation regiochemistry. The results indicate both that the oxidation regiochemistry is largely controlled by ionic interactions of the heme propionic acid groups with the protein and that shifts in regiospecificity involve rotation of the heme about an axis perpendicular to the heme plane.     

Probing the role of lysines and arginines in the catalytic function of cytochrome P450d by site-directed mutagenesis. Interaction with NADPH-cytochrome P450 reductase

To identify amino acids of cytochrome P450d (P450d) which participate in the interaction with NADPH-cytochrome P450 reductase, we changed conserved ionic amino acids of P450d to others by site-directed mutagenesis. Turnover numbers (0.032-0.008 min-1) of purified mutants Lys94-Glu, Lys99-Glu, Lys105-Glu, Lys440-Glu, Lys453-Glu, Arg455-Glu, and Lys463-Glu toward 7-ethoxycoumarin were much lower than that (0.380 min-1) of the wild type at 25 degrees C. Reduction rates (less than 0.054 s-1) of the heme of all mutants (0.1 microM) in the presence of NADPH and the reductase (0.3 microM) were much lower than that (5.9 s-1) of the wild type. Furthermore, a turnover number (0.042 min-1) of a microsomal triple mutant (Arg135-Leu + Arg136-Leu + Arg137-Leu) of a conserved Arg cluster was much lower than that (0.674 min-1) of the wild type at 37 degrees C. Thus, we suggest that Lys94, Lys99, Lys105, Lys440, Lys453, Arg455, Lys463, and perhaps the Arg cluster Arg135-Arg136-Arg137 of P450d will participate in the intermolecular electron transfer process by forming ionic bridges between the two proteins and/or by orienting appropriate geometry for electron transfer on the interfacial surface between the two proteins.     

Role of Positively Charged Residues Lys267, Lys270, and Arg411 of Cytochrome P450scc (Cyp11A1) in Interaction with Adrenodoxin

Cytochrome P450scc and adrenodoxin are redox proteins of the electron transfer chain of the inner mitochondrial membrane steroid hydroxylases. In the present work site-directed mutagenesis of the charged residues of cytochrome P450scc and adrenodoxin, which might be involved in interaction, was used to study the nature of electrostatic contacts between the hemeprotein and the ferredoxin. The target residues for mutagenesis were selected based on the theoretical model of cytochrome P450scc-adrenodoxin complex and previously reported chemical modification studies of cytochrome P450scc. In the present work, to clarify the molecular mechanism of hemeprotein interaction with ferredoxin, we constructed cytochrome P450scc Lys267, Lys270, and Arg411 mutants and Glu47 mutant of adrenodoxin and analyzed their possible role in electrostatic interaction and the role of these residues in the functional activity of the proteins. Charge neutralization at positions Lys267 or Lys270 of cytochrome P450scc causes no significant effect on the physicochemical and functional properties of cytochrome P450scc. However, cytochrome P450scc mutant Arg411Gln was found to exhibit decreased binding affinity to adrenodoxin and lower activity in the cholesterol side chain cleavage reaction. Studies of the functional properties of Glu47Gln and Glu47Arg adrenodoxin mutants indicate that a negatively charged residue in the loop covering the Fe2S2 cluster, being important for maintenance of the correct architecture of these structural elements of ferredoxin, is not directly involved in electrostatic interaction with cytochrome P450scc. Moreover, our results indicate the presence of at least two different binding (contact) sites on the proximal surface of cytochrome P450scc with different electrostatic input to interaction with adrenodoxin. In the binary complex, the positively charged sites of the proximal surface of cytochrome P450scc well correspond to the two negatively charged sites of adrenodoxin: the "interaction" domain site and the "core" domain site.     

A positively charged cluster formed in the heme-distal pocket of cytochrome P450nor is essential for interaction with NADH

Arg and Lys residues are concentrated on the distal side of cytochrome P450nor (P450nor) to form a positively charged cluster facing from the outside to the inside of the distal heme pocket. We constructed mutant proteins in which the Arg and Lys residues were replaced with Glu, Gln, or Ala. The results showed that this cluster plays crucial roles in NADH interaction. We also showed that some anions such as bromide (Br(-)) perturbed the heme environment along with the reduction step in P450nor-catalyzed reactions, which was similar to the effects caused by the mutations. We determined by x-ray crystallography that a Br(-), termed an anion hole, occupies a key region neighboring heme, which is the terminus of the positively charged cluster and the terminus of the hydrogen bond network that acts as a proton delivery system. A comparison of the predicted mechanisms between the perturbations caused by Br(-) and the mutations suggested that Arg(174) and Arg(64) play a crucial role in binding NADH to the protein. These results indicated that the positively charged cluster is the unique structure of P450nor that responds to direct interaction with NADH.     

Human heme oxygenase oxidation of 5- and 15-phenylhemes

Human heme oxygenase-1 (hHO-1) catalyzes the O2-dependent oxidation of heme to biliverdin, CO, and free iron. Previous work indicated that electrophilic addition of the terminal oxygen of the ferric hydroperoxo complex to the alpha-meso-carbon gives 5-hydroxyheme. Earlier efforts to block this reaction with a 5-methyl substituent failed, as the reaction still gave biliverdin IXalpha. Surprisingly, a 15-methyl substituent caused exclusive cleavage at the gamma-meso-rather than at the normal, unsubstituted alpha-meso-carbon. No CO was formed in these reactions, but the fragment cleaved from the porphyrin eluded identification. We report here that hHO-1 cleaves 5-phenylheme to biliverdin IXalpha and oxidizes 15-phenylheme at the alpha-meso position to give 10-phenylbiliverdin IXalpha. The fragment extruded in the oxidation of 5-phenylheme is benzoic acid, one oxygen of which comes from O2 and the other from water. The 2.29- and 2.11-A crystal structures of the hHO-1 complexes with 1- and 15-phenylheme, respectively, show clear electron density for both the 5- and 15-phenyl rings in both molecules of the asymmetric unit. The overall structure of 15-phenylheme-hHO-1 is similar to that of heme-hHO-1 except for small changes in distal residues 141-150 and in the proximal Lys18 and Lys22. In the 5-phenylheme-hHO-1 structure, the phenyl-substituted heme occupies the same position as heme in the heme-HO-1 complex but the 5-phenyl substituent disrupts the rigid hydrophobic wall of residues Met34, Phe214, and residues 26-42 near the alpha-meso carbon. The results provide independent support for an electrophilic oxidation mechanism and support a role for stereochemical control of the reaction regiospecificity.     

Role of LYS271 and LYS279 residues in the interaction of cytochrome P4501A1 with NADPH-cytochrome P450 reductase

It has been proposed that negatively charged amino acids on the surface of reductase and positively charged amino acids on the surface of P450 mediate the binding of both proteins through electrostatic interactions. In this study, we used a site-directed mutagenesis approach to determine a role for two lysine residues (Lys271 and Lys279) of cytochrome P4501A1 in the interaction of P4501A1 with reductase. We prepared two mutants P4501A1Ile271 and P4501A1Ile279 with a mutation of the lysine at positions 271 and 279, respectively. We observed a strong inhibition (>80%) of the 7-ethoxycoumarin and ethoxyresorufin deethylation activity in the reductase-supported system for both mutants. In the cumene hydroperoxide-supported system, P4501A1Ile279 exhibited wild-type activity, but the P4501A1Ile271 mutant activity remained low. The CD spectrum and substrate-binding assay indicated that the secondary structure of P4501A1Ile271 is perturbed. To evaluate further the involvement of these P4501A1 lysine residues in reductase binding, we measured the KM of reductase for wild type and mutants. Both wild type and P4501A1Ile271 reached saturation in the range of reductase concentrations tested with KM values 5.1 and 11.2 pM, respectively. The calculated KM value for P4501A1Ile279 increased 9-fold, 44.4 pM, suggesting that the mutation affected binding of reductase to P4501A1. Stopped-flow spectroscopy was employed to evaluate the effect of mutations on electron transfer from reductase to heme iron. Both wild type and P450Ile279 showed biphasic kinetics with a approximately 40% participation of the fast step in the total activity. On the other hand, only single-phase kinetics for iron reduction was observed for P450Ile271, suggesting that the low activity of this mutant can be attributed not only to major structural changes but also to a disturbance in the electron transport.     

Fungal heme oxygenases: Functional expression and characterization of Hmx1 from Saccharomyces cerevisiae and CaHmx1 from Candida albicans

Heme oxygenases convert heme to free iron, CO, and biliverdin. Saccharomyces cerevisiae and Candida albicans express putative heme oxygenases that are required for the acquisition of iron from heme, a critical process for fungal survival and virulence. The putative heme oxygenases Hmx1 and CaHmx1 from S. cerevisiae and C. albicans, respectively, minus the sequences coding for C-terminal membrane-binding domains, have been expressed in Escherichia coli. The C-terminal His-tagged, truncated enzymes are obtained as soluble, active proteins. Purified ferric Hmx1 and CaHmx1 have Soret absorption maxima at 404 and 410 nm, respectively. The apparent heme binding Kd values for Hmx1 and CaHmx1 are 0.34 +/- 0.09 microM and 1.0 +/- 0.2 microM, respectively. The resonance Raman spectra of Hmx1 reveal a heme binding pocket similar to those of the mammalian and bacterial heme oxygenases. Several reductants, including ascorbate, yeast cytochrome P450 reductase (CPR), human CPR, spinach ferredoxin/ferredoxin reductase, and putidaredoxin/putidaredoxin reductase, are able to provide electrons for biliverdin production by Hmx1 and CaHmx1. Of these, ascorbate is the most effective reducing partner. Heme oxidation by Hmx1 and CaHmx1 regiospecifically produces biliverdin IXalpha. Spectroscopic analysis of aerobic reactions with H2O2 identifies verdoheme as a reaction intermediate. Hmx1 and CaHmx1 are the first fungal heme oxygenases to be heterologously overexpressed and characterized. Their heme degradation activity is consistent with a role in iron acquisition.     

Evolutionarily divergent electron donor proteins interact with P450MT2 through the same helical domain but different contact points

We have investigated the sites of N-terminally truncated cytochrome P4501A1 targeted to mitochondria (P450MT2) which interact with adrenodoxin (Adx), cytochrome P450 reductase (CPR) and bacterial flavodoxin (Fln). The binding site was mapped by a combination of in vitro mutagenesis, in vivo screening with a mammalian two-hybrid system, spectral analysis, reconstitution of enzyme activity and homology-based structural modeling. Our results show that part of an aqueous accessible helix (putative helix G, residues 264-279) interacts with all three electron donor proteins. Mutational studies revealed that Lys267 and Lys271 are crucial for binding to Adx, while Lys268 and Arg275 are important for binding to CPR and FLN: Additive effects of different electron donor proteins on enzyme activity and models on protein docking show that Adx and CPR bind in a non-overlapping manner to the same helical domain in P450MT2 at different angular orientations, while CPR and Fln compete for the same binding site. We demonstrate that evolutionarily divergent electron donor proteins interact with the same domain but subtly different contact points of P450MT2.     

Interaction of nitric oxide with human heme oxygenase-1

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

Expression and characterization of full-length human heme oxygenase-1: the presence of intact membrane-binding region leads to increased binding affinity for NADPH cytochrome P450 reductase

Heme oxygenase-1 (HO-1) is the chief regulatory enzyme in the oxidative degradation of heme to biliverdin. In the process of heme degradation, HO-1 receives the electrons necessary for catalysis from the flavoprotein NADPH cytochrome P450 reductase (CPR), releasing free iron and carbon monoxide. Much of the recent research involving heme oxygenase has been done using a 30 kDa soluble form of the enzyme, which lacks the membrane binding region (C-terminal 23 amino acids). The goal of this study was to express and purify a full-length human HO-1 (hHO-1) protein; however, due to the lability of the full-length form, a rapid purification procedure was required. This was accomplished by use of a glutathione-s-transferase (GST)-tagged hHO-1 construct. Although the procedure permitted the generation of a full-length HO-1, this form was contaminated with a 30 kDa degradation product that could not be eliminated. Therefore, attempts were made to remove a putative secondary thrombin cleavage site by a conservative mutation of amino acid 254, which replaces arginine with lysine. This mutation allowed the expression and purification of a full-length hHO-1 protein. Unlike wild type (WT) HO-1, the R254K mutant could be purified to a single 32 kDa protein capable of degrading heme at the same rate as the WT enzyme. The R254K full-length form had a specific activity of approximately 200-225 nmol of bilirubin h-1 nmol-1 HO-1 as compared to approximately 140-150 nmol of bilirubin h-1 nmol-1 for the WT form, which contains the 30 kDa contaminant. This is a 2-3-fold increase from the previously reported soluble 30 kDa HO-1, suggesting that the C-terminal 23 amino acids are essential for maximal catalytic activity. Because the membrane-spanning domain is present, the full-length hHO-1 has the potential to incorporate into phospholipid membranes, which can be reconstituted at known concentrations, in combination with other endoplasmic reticulum resident enzymes.     

Molecular mechanism of the electron transfer reaction in cytochrome P450(cam)--putidaredoxin: roles of glutamine 360 at the heme proximal site

We characterized electron transfer (ET) from putidaredoxin (Pdx) to the mutants of cytochrome P450(cam) (P450(cam)), in which one of the residues located on the putative binding site to Pdx, Gln360, was replaced with Glu, Lys, and Leu. The kinetic analysis of the ET reactions from reduced Pdx to ferric P450(cam) (the first ET) and to ferrous oxygenated P450(cam) (the second ET) showed the dissociation constants (K(m)) that were moderately perturbed for the Lys and Leu mutants and the distinctly increased for the Glu mutant. Although the alterations in K(m) indicate that Gln360 is located at the Pdx binding site, the effects of the Gln360 mutations (0.66-20-fold of that of wild type) are smaller than those of the Arg112 mutants (25-2500-fold of that of wild type) [Unno, M., et al. (1996) J. Biol. Chem. 271, 17869-17874], allowing us to conclude that Gln360 much less contributes to the complexation with Pdx than Arg112. The first ET rate (35 s(-1) for wild-type P450(cam)) was substantially reduced in the Glu mutant (5.4 s(-1)), while less perturbation was observed for the Lys (53 s(-1)) and Leu (23 s(-1)) mutants. In the second ET reaction, the retarded ET rate was detected only in the Glu mutant but not in the Lys and Leu mutants. These results showed the smaller mutational effects of Gln360 on the ET reactions than those of the Arg112 mutants. In contrast to the moderate perturbations in the kinetic parameters, the mutations at Gln360 significantly affected both the standard enthalpy and entropy of the redox reaction of P450(cam), which cause the negative shift of the redox potentials for the Fe(3+)/Fe(2+) couple by 20-70 mV. Since the amide group of Gln360 is located near the carbonyl oxygen of the amide group of the axial cysteine, it is plausible that the mutation at Gln360 perturbs the electronic interaction of the axial ligand with heme iron, resulting in the reduction of the redox potentials. We, therefore, conclude that Gln360 primarily regulates the ET reaction of P450(cam) by modulating the redox potential of the heme iron and not by the specific interaction with Pdx or the formation of the ET pathway that are proposed as the regulation mechanism of Arg112.     

Engineering of proteolytically stable NADPH-cytochrome P450 reductase

NADPH-cytochrome P450 reductase (CPR) is a membrane-bound flavoprotein that interacts with the membrane via its N-terminal hydrophobic sequence (residues 1-56). CPR is the main electron transfer component of hydroxylation reactions catalyzed by microsomal cytochrome P450s. The membrane-bound hydrophobic domain of NADPH-cytochrome P450 reductase is easily removed during limited proteolysis and is the subject of spontaneous digestion of membrane-binding fragment at the site Lys56-Ile57 by intracellular trypsin-like proteases that makes the flavoprotein very unstable during purification or expression in E. coli. The removal of the N-terminal hydrophobic sequence of NADPH-cytochrome P450 reductase results in loss of the ability of the flavoprotein to interact and transfer electrons to cytochrome P450. In the present work, by replacement of the lysine residue (Lys56) with Gln using site directed mutagenesis, we prepared the full-length flavoprotein mutant Lys56Gln stable to spontaneous proteolysis but possessing spectral and catalytic properties of the wild type flavoprotein. Limited proteolysis with trypsin and protease from Staphylococcus aureus of highly purified and membrane-bound Lys56Gln mutant of the flavoprotein as well as wild type NADPH-cytochrome P450 reductase allowed localization of some amino acids of the linker fragment of NADPH-cytochrome P450 reductase relative to the membrane. During prolong incubation or with increased trypsin ratio, the mutant form showed an alternative limited proteolysis pattern, indicating the partial accessibility of another site. Nevertheless, the membrane-bound mutant form is stable to trypsinolysis. Truncated forms of the flavoprotein (residues 46-676 of the mutant or 57-676 of wild type NADPH-cytochrome P450 reductase) are unable to transfer electrons to cytochrome P450c17 or P4503A4, confirming the importance of the N-terminal sequence for catalysis. Based on the results obtained in the present work, we suggest a scheme of structural topology of the N-terminal hydrophobic sequence of NADPH-cytochrome P450 reductase in the membrane.     

The maintenance of high affinity plasminogen binding by group A streptococcal plasminogen-binding M-like protein is mediated by arginine and histidine residues within the a1 and a2 repeat domains

Subversion of the plasminogen activation system is implicated in the virulence of group A streptococci (GAS). GAS displays receptors for the human zymogen plasminogen on the cell surface, one of which is the plasminogen-binding group A streptococcal M-like protein (PAM). The plasminogen binding domain of PAM is highly variable, and this variation has been linked to host selective immune pressure. Site-directed mutagenesis of full-length PAM protein from an invasive GAS isolate was undertaken to assess the contribution of residues in the a1 and a2 repeat domains to plasminogen binding function. Mutagenesis to alanine of key plasminogen binding lysine residues in the a1 and a2 repeats (Lys98 and Lys111) did not abrogate plasminogen binding by PAM nor did additional mutagenesis of Arg101 and His102 and Glu104, which have previously been implicated in plasminogen binding. Plasminogen binding was only abolished with the additional mutagenesis of Arg114 and His115 to alanine. Furthermore, mutagenesis of both arginine (Arg101 and Arg114) and histidine (His102 and His115) residues abolished interaction with plasminogen despite the presence of Lys98 and Lys111 in the binding repeats. This study shows for the first time that residues Arg101, Arg114, His102, and His115 in both the a1 and a2 repeat domains of PAM can mediate high affinity plasminogen binding. These data suggest that highly conserved arginine and histidine residues may compensate for variation elsewhere in the a1 and a2 plasminogen binding repeats, and may explain the maintenance of high affinity plasminogen binding by naturally occurring variants of PAM.     

Covalently crosslinked complexes of bovine adrenodoxin with adrenodoxin reductase and cytochrome P450scc. Mass spectrometry and Edman degradation of complexes of the steroidogenic hydroxylase system.

NADPH-dependent adrenodoxin reductase, adrenodoxin and several diverse cytochromes P450 constitute the mitochondrial steroid hydroxylase system of vertebrates. During the reaction cycle, adrenodoxin transfers electrons from the FAD of adrenodoxin reductase to the heme iron of the catalytically active cytochrome P450 (P450scc). A shuttle model for adrenodoxin or an organized cluster model of all three components has been discussed to explain electron transfer from adrenodoxin reductase to P450. Here, we characterize new covalent, zero-length crosslinks mediated by 1-ethyl-3-(3-dimethylaminopropyl) carbodiimide between bovine adrenodoxin and adrenodoxin reductase, and between adrenodoxin and P450scc, respectively, which allow to discriminate between the electron transfer models. Using Edman degradation, mass spectrometry and X-ray crystallography a crosslink between adrenodoxin reductase Lys27 and adrenodoxin Asp39 was detected, establishing a secondary polar interaction site between both molecules. No crosslink exists in the primary polar interaction site around the acidic residues Asp76 to Asp79 of adrenodoxin. However, in a covalent complex of adrenodoxin and P450scc, adrenodoxin Asp79 is involved in a crosslink to Lys403 of P450scc. No steroidogenic hydroxylase activity could be detected in an adrenodoxin -P450scc complex/adrenodoxin reductase test system. Because the acidic residues Asp76 and Asp79 belong to the binding site of adrenodoxin to adrenodoxin reductase, as well as to the P450scc, the covalent bond within the adrenodoxin-P450scc complex prevents electron transfer by a putative shuttle mechanism. Thus, chemical crosslinking provides evidence favoring the shuttle model over the cluster model for the steroid hydroxylase system.     

Localization of von willebrand factor-binding sites for platelet glycoprotein Ib and botrocetin by charged-to-alanine scanning mutagenesis

At sites of vascular injury, von Willebrand factor (VWF) mediates platelet adhesion through binding to platelet glycoprotein Ib (GPIb). Previous studies identified clusters of charged residues within VWF domain A1 that were involved in binding GPIb or botrocetin. The contribution of 28 specific residues within these clusters was analyzed by mutating single amino acids to alanine. Binding to a panel of six conformation-dependent monoclonal antibodies was decreased by mutations at Asp(514), Asp(520), Arg(552), and Arg(611) (numbered from the N-terminal Ser of the mature processed VWF), suggesting that these residues are necessary for domain A1 folding. Binding of (125)I-botrocetin was decreased by mutations at Arg(629), Arg(632), Arg(636), and Lys(667). Ristocetin-induced and botrocetin-induced binding to GPIb both were decreased by mutations at Lys(599), Arg(629), and Arg(632); among this group the K599A mutant was unique because (125)I-botrocetin binding was normal, suggesting that Lys(599) interacts directly with GPIb. Ristocetin and botrocetin actions on VWF were dissociated readily by mutagenesis. Ristocetin-induced binding to GPIb was reduced selectively by substitutions at positions Lys(534), Arg(571), Lys(572), Glu(596), Glu(613), Arg(616), Glu(626), and Lys(642), whereas botrocetin-induced binding to GPIb was decreased selectively by mutations at Arg(636) and Lys(667). The binding of monoclonal antibody B724 involved Lys(660) and Arg(663), and this antibody inhibits (125)I-botrocetin binding to VWF. The crystal structure of the A1 domain suggests that the botrocetin-binding site overlaps the monoclonal antibody B724 epitope on helix 5 and spans helices 4 and 5. The binding of botrocetin also activates the nearby VWF-binding site for GPIb that involves Lys(599) on helix 3.     

Molecular characterization of an extended binding site for coagulation factor Va in the positive exosite of activated protein C

The anticoagulant human plasma serine protease, activated protein C (APC), inhibits blood coagulation by specific inactivation of the coagulation cofactors factor Va (FVa) and factor VIIIa. Site-directed mutagenesis of residues in three surface loops of a positive exosite located on APC was used to identify residues that play a significant role in binding to FVa. Eighteen different residues were mutated to alanine singly, in pairs, or in triple mutation combinations. Mutant APC proteins were purified and characterized for their inactivation of FVa. Three APC residues were identified that provide major contributions to FVa interactions: Lys(193), Arg(229), and Arg(230). In addition, four residues made significant minor contributions to FVa interactions: Lys(191), Lys(192), Asp(214), and Glu(215). All of these residues primarily contribute to APC cleavage at Arg(506) in FVa and play a small role in the interaction of APC with the Arg(306) cleavage site. In conjunction with previously published work, these results define an extensive FVa binding site in the positive exosite of APC that is primarily involved in binding and cleaving at Arg(506) on FVa.     

Site-Directed Mutagenesis of Cytochrome P450scc (CYP11A1). Effect of Lysine Residue Substitution on Its Structural and Functional Properties

Our previous chemical modification and cross-linking studies identified some positively charged amino acid residues of cytochrome P450scc that may be important for its interaction with adrenodoxin and for its functional activity. The present study was undertaken to further evaluate the role of these residues in the interaction of cytochrome P450scc with adrenodoxin using site-directed mutagenesis. Six cytochrome P450scc mutants containing replacements of the surface-exposed positively charged residues (Lys103Gln, Lys110Gln, Lys145Gln, Lys394Gln, Lys403Gln, and Lys405Gln) were expressed in E. coli cells, purified as a substrate-bound high-spin form, and characterized as compared to the wild-type protein. The replacement of the surface Lys residues does not dramatically change the protein folding or the heme pocket environment as judged from limited proteolysis and spectral studies of the cytochrome P450 mutants. The replacement of Lys in the N-terminal sequence of P450scc does not dramatically affect the activity of the heme protein. However, mutant Lys405Gln revealed rather dramatic loss of cholesterol side-chain cleavage activity, efficiency of enzymatic reduction in a reconstituted system, and apparent dissociation constant for adrenodoxin binding. The present results, together with previous findings, suggest that the changes in functional activity of mutant Lys405Gln may reflect the direct participation of this amino acid residue in the electrostatic interaction of cytochrome P450scc with its physiological partner, adrenodoxin.     

Biochemical characterization of rat P450 2C11 fused to rat or bacterial NADPH-P450 reductase domains

cDNAs coding for rat P450 2C11 fused to either a bacterial (the NADPH-cytochrome P450 BM3 reductase domain of P450 BM3) or a truncated form of rat NADPH-P450 reductases were expressed in Escherichia coli and characterized enzymatically. Measurements of NADPH cytochrome c reductase activity showed fusion-dependent increases in the rates of cytochrome c reduction by the bacterial or the mammalian flavoprotein (21 and 48%, respectively, of the rates observed with nonfused enzymes). Neither the bacterial flavoprotein nor the truncated rat reductase supported arachidonic acid metabolism by P450 2C11. In contrast, fusion of P450 2C11 to either reductase yielded proteins that metabolized arachidonic acid to products similar to those obtained with reconstituted systems containing P450 2C11 and native rat P450 reductase. Addition of a 10-fold molar excess of rat P450 reductase markedly increased the rates of metabolism by both fused and nonfused P450s 2C11. These increases occurred with preservation of the regioselectivity of arachidonic acid metabolism. The fusion-independent reduction of P450 2C11 by bacterial P450 BM3 reductase was shown by measurements of NADPH-dependent H(2)O(2) formation [73 +/- 10 and 10 +/- 1 nmol of H(2)O(2) formed min(-)(1) (nmol of P450)(-)(1) for the reconstituted and fused protein systems, respectively]. These studies demonstrate that (a) a self-sufficient, catalytically active arachidonate epoxygenase can be constructed by fusing P450 2C11 to mammalian or bacterial P450 reductases and (b) the P450 BM3 reductase interacts efficiently with mammalian P450 2C11 and catalyzes the reduction of the heme iron. However, fusion is required for metabolism and product formation.     

Effect of mutations at Lys250, Arg251, and Lys253 of cytochrome P450 1A2 on the catalytic activities and the bindings of bifunctional axial ligands.

Some eukaryotic cytochromes P450 (P450s) have a series of ionic amino acids, corresponding to Lys250, Arg251, and Lys253 residues in the P450 1A2 sequence. To understand the roles of those ionic amino acids in the catalytic function of P450, three single mutants, Lys250Leu, Arg251Leu, and Lys253Leu of P450 1A2 were obtained from yeast (Saccharomyces cerevisiae) expression system. Turnover numbers of the Arg251Leu mutant in dealkylation reactions of methoxy- and ethoxyresorufin catalyzed by the P450 reconstituted system were remarkably increased by sixfold compared to those of the wild type. The Lys250Leu and Lys253Leu mutants also showed turnover numbers higher than those of the wild type by three- to fourfold. Those catalytic activities were inhibited competitively by pyridine derivatives, nitrogenous axial ligands to the P450 heme. From those findings together with other spectral data, it was suggested that the ionic site of Lys250, Arg251, and Lys253 may be somehow located near the substrate recognition site and/or near the axial-ligand access channel of this enzyme.