'I feel like a person has a right to use a product to protect themselves ': a qualitative study of the risk-benefit calculus on women's contraceptive use and choice.

Background Reducing pregnancy risk requires a multidimensional approach to sexual and reproductive health product development. The purpose of this analysis is to identify, compare, and contrast women's pre-use beliefs and attitudes about three different forms of contraceptives: intravaginal rings; spermicide in conjunction with condoms; and oral contraceptive pills - and explore how those attitudes and beliefs, along with actual method-use experience, may affect potential choices in contraceptive method moving forward. The relationship of beliefs and attitudes to their risk-benefit calculations when using these methods was also considered.? METHODS: Women used one or more contraceptive methods, each for 3-6 months. Qualitative data from individual in-depth interviews completed after each 3-month use period were analysed using a summary matrix framework. Data were extracted and summarised into themes. Each woman's experiences were compared among the methods she used; comparisons were also made across participants. RESULTS: The data consist of 33 90-120 min in-depth qualitative interviews from 16 women aged 20-34 years, in which they discussed various elements of their method use experience. One prominent theme was identified: the influence of attitudes and beliefs on the risk-benefit calculus. There were six key elements within the theme: pregnancy prevention; dosing and the potential for user error; side-effects; familiarity; disclosure; and sexual partnerships. CONCLUSIONS: Women weighed perceived risks and benefits in their decision-making and, ultimately, their contraception choices. Understanding women's beliefs and attitudes that contribute to a calculation of risk-benefit can inform the development of sexual and reproductive health products.

A Qualitative Study of the Contraceptive Effect on Women's Sexual Experiences: Beyond Hormonal Effects.

OBJECTIVE: To elucidate the effects of the intravaginal ring, oral contraceptive pill (OCP), and spermicide plus condom on women's sexual experiences through an in-depth understanding of the physical characteristics of these contraceptive methods. METHODS: We conducted qualitative in-depth interviews with women (aged 18-45 years) who used up to three contraceptive methods (intravaginal ring, OCP, and spermicide plus condom). Women completed in-depth interviews after each 3-month use period. We used a summarized matrix framework and thematic content analysis to explore how each method affected participants' sexual experiences. RESULTS: Sixteen women completed interviews, yielding 33 transcripts. Women reported physical effects on their sexual experiences while using the intravaginal ring and spermicide plus condom. The OCP was often discussed as lacking these physical effects. Discussion themes included product administration (eg, navigating intravaginal ring removal) and physical product awareness (eg, spermicide as a lubricant). From these experiences, women often altered and individualized their use and subsequent opinions of the contraceptive method. CONCLUSION: The range of contraceptive effects on women's sexual experiences shape their use and opinions of the product, leading to either increased motivation and consistent use or poor adherence and discontinuation. Awareness of these individualized experiences can help providers better understand and guide their patients towards successful contraceptive use.

NMR studies of the dynamics of high-spin nitrophorins: comparative studies of NP4 and NP2 at close to physiological pH.

The ß-barrel nitrophorin (NP) heme proteins are found in the saliva of the blood-sucking insect Rhodnius prolixus, which synthesizes and stores nitric oxide (NO) in the salivary glands. NO is bound to iron of the NPs and is released by dilution and an increase in pH when the insect spits its saliva into the tissues of a victim, to aid in obtaining a blood meal. In the adult insect, there are four nitrophorins, NP1-NP4, which have sequence similarities in two pairs, NP1 and NP4 (90% identical) and NP2 and NP3 (80% identical). The available crystal structures of NP4 have been used to propose that pH-dependent changes in the conformation of two loops between adjacent ß-strands at the front opening of the protein, the A-B and G-H loops, determine the rate of NO release. At pH 7.3, NP4 releases NO 17 times faster than NP2 does. In this work, the aqua complexes of NP4 and NP2 have been investigated by nuclear magnetic resonance (NMR) relaxation measurements to probe the pico- to nanosecond and micro- to millisecond time scale motions at two pH values, 6.5 and 7.3. It is found that NP4-OH2 is fairly rigid and only residues in the loop regions show dynamics at pH 6.5; at pH 7.3, much more dynamics of the loops and most of the ß-strands are observed while the α-helices remain fairly rigid. In comparison, NP2-OH2 shows much less dynamics, albeit somewhat more than that of the previously reported NP2-NO complex [Muthu, D., Berry, R. E., Zhang, H., and Walker, F. A. (2013) Biochemistry 52, 7910-7925]. The reasons for this major difference between NP4 and NP2 are discussed.

Dimerization of nitrophorin 4 at low pH and comparison to the K1A mutant of nitrophorin 1.

Nitrophorin 4, one of the four NO-carrying heme proteins from the salivary glands of Rhodnius prolixus, forms a homodimer at pH 5.0 with a Kd of ∼8 µM. This dimer begins to dissociate at pH 5.5 and is completely dissociated to monomer at pH 7.3, even at 3.7 mM. The dimer is significantly stabilized by binding NO to the heme and at pH 7.3 would require dilution to well below 0.2 mM to completely dissociate the NP4-NO homodimer. The primary techniques used for investigating the homodimer and the monomer-dimer equilibrium were size-exclusion fast protein liquid chromatography at pH 5.0 and (1)H{(15)N} heteronuclear single-quantum coherence spectroscopy as a function of pH and concentration. Preparation of site-directed mutants of NP4 (A1K, D30A, D30N, V36A/D129A/L130A, K38A, R39A, K125A, K125E, D132A, L133V, and K38Q/R39Q/K125Q) showed that the N-terminus, D30, D129, D132, at least one heme propionate, and, by association, likely also E32 and D35 are involved in the dimerization. The "closed loop" form of the A-B and G-H flexible loops of monomeric NP4, which predominates in crystal structures of the monomeric protein reported at pH 5.6 but not at pH 7.5 and which involves all of the residues listed above except D132, is required for dimer formation. Wild-type NP1 does not form a homodimer, but NP1(K1A) and native N-terminal NP1 form dimers in the presence of NO. The homodimer of NP1, however, is considerably less stable than that of NP4 in the absence of NO. This suggests that additional aspartate or glutamate residues present in the C-terminal region of NP4, but not NP1, are also involved in stabilizing the dimer.

NMR investigations of nitrophorin 2 belt side chain effects on heme orientation and seating of native N-terminus NP2 and NP2(D1A).

Nitrophorin 2 (NP2), one of the four NO-storing and NO-releasing proteins found in the saliva of the blood-sucking bug Rhodnius prolixus, has a more ruffled heme and a high preference for a particular heme orientation (B) compared with nitrophorin 1 and nitrophorin 4, which show not a preference (A to B ratio of approximately 1:1), suggesting that it fits more tightly in the ß-barrel protein. In this work we have prepared a series of "belt" mutants of NP2(D1A) and (ΔM0)NP2 aimed at reducing the size of aromatic or other residues that surround the heme, and investigated them as the high-spin aqua and low-spin N-methylimidazole complexes. The belt mutants included Y38A, Y38F, F42A, F66A, Y85A, Y85F, Y104A, I120T, and a triple mutant of NP2(D1A), the F42L, L106F, I120T mutant. Although I120 has been mainly considered to be a distal pocket residue, CδH3 of I120 lies directly above the heme 3-methyl, at 2.67 Å, of heme orientation B, or the 2-vinyl of A, and it thus plays a role as a belt mutant, a role that turns out to be extremely important in creating the strong favoring of the B heme orientation [A to B ratio of 1:14 for NP2(D1A) or 1:12 for (ΔM0)NP2]. The results show that the 1D (1)H NMR spectra of the high-spin forms are quite sensitive to changes in the shape of the heme binding cavity. The single mutation I120T eliminates the favorability of the B heme orientation by producing a heme A to B orientation ratio of 1:1, whereas the single mutation F42A reverses the heme orientation from an A to B ratio of 1:14 seen for NP2(D1A) to 10:1 for NP2(D1A,F42A). The most extreme ratio was found for the triple mutant of NP2(D1A), NP2(D1A,F42L,L105F,I120T), in which the A to B ratio is approximately 25:1, a ΔG change of about -3.5 kcal/mol or -14.1 kJ/mol with respect to NP2(D1A). The seating of the heme is modified as well in that mutant and in several others, by rotations of the heme by up to 4° from the seating observed in NP2(D1A), in order to relieve steric interactions between a vinyl ß-carbon and a protein side chain, or to fill a cavity created by replacing a large protein side chain by a much smaller one; the latter was observed for all tyrosine to alanine mutants. These relatively small changes in seating have a measurable effect on the NMR spectra of the mutants, but are indeed minor in terms of overall seating and reactivity of the NP2(D1A) protein. The (1)H NMR resonances of the hemin substituents of the low-spin N-methylimidazole complexes of NP2(D1A,F42L,L105F,I120T) as well as NP2(D1A,I120T), NP2(D1A,Y104A), and NP2(D1A,F42A) have been assigned using natural abundance (1)H{(13)C} heteronuclear multiple quantum correlation and (1)H-(1)H nuclear Overhauser effect spectroscopy spectra.

Institute of medicine 2009 gestational weight gain guideline knowledge: survey of obstetrics/gynecology and family medicine residents of the United States.

BACKGROUND: In 2009, the Institute of Medicine revised gestational weight gain recommendations; revisions included body mass index (BMI) category cut-point changes and provision of range of gain for obese women. Our objective was to examine resident prenatal caregivers' knowledge of revised guidelines. METHODS: Anonymous electronic survey of obstetrics/gynecology and family medicine residents across the United States from January to April 2010. RESULTS: Overall, 660 completed the survey; 79 percent female and 69 percent aged between 21 and 30. When permitted to select ≥ 1 response, 87.0 percent reported using BMI to assess weight status at initial visits, 44.4 percent reported using "clinical impression based on patient appearance," and 1.4 percent reported not using any parameters. When asked the most important baseline parameter for providing recommendations, 35.8 percent correctly identified prepregnancy BMI, 2.1 percent reported "I don't provide guidelines," and 4.5 percent reported "I do not discuss gestational weight gain." Among respondents, 57.6 percent reported not being aware of new guidelines. Only 7.6 percent selected correct BMI ranges for each category, and only 5.8 percent selected correct gestational weight gain ranges. Only 2.3 percent correctly identified both BMI cutoffs and recommended gestational weight gain ranges per 2009 guidelines. CONCLUSIONS: Guideline knowledge is the foundation of accurate counseling, yet resident prenatal caregivers were minimally aware of the 2009 Institute of Medicine gestational weight gain guidelines almost a year after their publication.

NMR studies of the dynamics of nitrophorin 2 bound to nitric oxide.

The Rhodnius nitrophorins are ß-barrel proteins of the lipocalin fold with a heme protruding from the open end of the barrel. They are found in the saliva of the blood-sucking insect Rhodnius prolixus, which synthesizes and stores nitric oxide (NO) in the salivary glands, where NO is bound to iron. NO is released by dilution and an increase in pH when the insect spits its saliva into the tissues of a victim, to aid in obtaining a blood meal. In the adult insect, there are four nitrophorins, NP1-NP4. At pH 7.3, NP4 releases NO 17 times faster than NP2 does, as measured by stopped-flow kinetics. A number of crystal structures of the least abundant protein, NP4, are available. These structures have been used to propose that two loops between adjacent ß-strands at the front opening of the protein, the A-B and G-H loops, determine the rate of NO release. To learn how the protein loops contribute to the release of NO for each of the nitrophorins, the dynamics of these proteins are being studied in our laboratory. In this work, the NP2-NO complex has been investigated by nuclear magnetic resonance relaxation measurements to probe the picosecond-to-nanosecond and microsecond-to-millisecond time scale motions at three pH values, 5.0, 6.5, and 7.3. It is found that at pH 5.0 and 6.5, the NP2-NO complex is rigid and only a few residues in the loop regions show dynamics, while at pH 7.3, somewhat more dynamics, particularly of the A-B loop, are observed. Comparison to other lipocalins shows that all are relatively rigid, and that the dynamics of lipocalins in general are much more subtle than those of mainly α-helical proteins.

Probing the role of a conserved salt bridge in the intramolecular electron transfer kinetics of human sulfite oxidase.

Sulfite oxidase (SO) is a vital metabolic enzyme that catalyzes the oxidation of toxic sulfite to sulfate. The proposed mechanism of this molybdenum cofactor dependent enzyme involves two one-electron intramolecular electron transfer (IET) steps from the molybdenum center to the iron of the b 5-type heme and two one-electron intermolecular electron transfer steps from the heme to cytochrome c. This work focuses on how the electrostatic interaction between two conserved amino acid residues, R472 and D342, in human SO (hSO) affects catalysis. The hSO variants R472M, R472Q, R472K, R472D, and D342K were created to probe the effect of the position of the salt bridge charges, along with the interaction between these two residues. With the exception of R472K, these variants all showed a significant decrease in their IET rate constants, k et, relative to wild-type hSO, indicating that the salt bridge between residues 472 and 342 is important for rapid IET. Surprisingly, however, except for R472K and R472D, all of the variants show k cat values higher than their corresponding k et values. The turnover number for R472D is about the same as k et, which suggests that the change in this variant is rate-limiting in catalysis. Direct spectroelectrochemical determination of the Fe(III/II) reduction potentials of the heme and calculation of the Mo(VI/V) potentials revealed that all of the variants affected the redox potentials of both metal centers, probably due to changes in their environments. Thus, the position of the positive charge of R472 and that of the negative charge of D342 are both important in hSO, and changing either the position or the nature of these charges perturbs IET and catalysis.

Electron spin density on the axial His ligand of high-spin and low-spin nitrophorin 2 probed by heteronuclear NMR spectroscopy.

The electronic structure of heme proteins is exquisitely tuned by the interaction of the iron center with the axial ligands. NMR studies of paramagnetic heme systems have been focused on the heme signals, but signals from the axial ligands have been rather difficult to detect and assign. We report an extensive assignment of the (1)H, (13)C and (15)N resonances of the axial His ligand in the NO-carrying protein nitrophorin 2 (NP2) in the paramagnetic high-spin and low-spin forms, as well as in the diamagnetic NO complex. We find that the high-spin protein has σ spin delocalization to all atoms in the axial His57, which decreases in size as the number of bonds between Fe(III) and the atom in question increases, except that within the His57 imidazole ring the contact shifts are a balance between positive σ and negative π contributions. In contrast, the low-spin protein has π spin delocalization to all atoms of the imidazole ring. Our strategy, adequately combined with a selective residue labeling scheme, represents a straightforward characterization of the electron spin density in heme axial ligands.

Effects of mutating aromatic surface residues of the heme domain of human sulfite oxidase on its heme midpoint potential, intramolecular electron transfer, and steady-state kinetics.

Human sulfite oxidase (hSO), an essential molybdoheme enzyme, catalyzes the oxidation of toxic sulfite to sulfate. The proposed catalytic cycle includes two, one-electron intramolecular electron transfers (IET) between the molybdenum (Mo) and the heme domains. Rapid IET rates are ascribed to conformational changes that bring the two domains into close proximity to one another. Previous studies of hSO have focused on the roles of conserved residues near the Mo active site and on the tether that links the two domains. Here four aromatic surface residues on the heme domain (phenylalanine 57 (F57), phenylalanine 79 (F79), tyrosine 83 (Y83), and histidine 90 (H90)) have been mutated, and their involvement in IET rates, the heme midpoint potential, and the catalytic activity of hSO have been investigated using laser flash photolysis, spectroelectrochemistry, and steady-state kinetics, respectively. The results indicate that the size and hydrophobicity of F57 play an important role in modulating the heme potential and that F57 also affects the IET rates. The data also suggest that important interactions of H90 with a heme propionate group destabilize the Fe(III) state of the heme. The positive charge on H90 at pH ≤ 7.0 may decrease the electrostatic interaction between the Mo and heme domains, thereby decreasing the IET rates of wt hSO at low pH. Lastly, mutations of F79 and Y83, which are located on the surface of the heme domain, but not in direct contact with the heme or the propionate groups, have little effect on either IET or the heme potential.

Native N-terminus nitrophorin 2 from the kissing bug: similarities to and differences from NP2(D1A).

The first amino acid of mature native nitrophorin 2 is aspartic acid, and when expressed in E. coli, the wild-type gene of the mature protein retains the methionine-0, which is produced by translation of the start codon. This form of NP2, (M0)NP2, has been found to have different properties from its D1A mutant, for which the Met0 is cleaved by the methionine aminopeptidase of E. coli (R. E. Berry, T. K. Shokhireva, I. Filippov, M. N. Shokhirev, H. Zhang, F. A. Walker, Biochemistry 2007, 46, 6830). Native N-terminus nitrophorin 2 ((ΔM0)NP2) has been prepared by employing periplasmic expression of NP2 in E. coli using the pelB leader sequence from Erwinia carotovora, which is present in the pET-26b expression plasmid (Novagen). This paper details the similarities and differences between the three different N-terminal forms of nitrophorin 2, (M0)NP2, NP2(D1A), and (ΔM0)NP2. It is found that the NMR spectra of high- and low-spin (ΔM0)NP2 are essentially identical to those of NP2(D1A), but the rate and equilibrium constants for histamine and NO dissociation/association of the two are different.

Nuclear inelastic scattering and Mössbauer spectroscopy as local probes for ligand binding modes and electronic properties in proteins: vibrational behavior of a ferriheme center inside a ß-barrel protein.

In this work, we present a study of the influence of the protein matrix on its ability to tune the binding of small ligands such as NO, cyanide (CN(-)), and histamine to the ferric heme iron center in the NO-storage and -transport protein Nitrophorin 2 (NP2) from the salivary glands of the blood-sucking insect Rhodnius prolixus. Conventional Mössbauer spectroscopy shows a diamagnetic ground state of the NP2-NO complex and Type I and II electronic ground states of the NP2-CN(-) and NP2-histamine complex, respectively. The change in the vibrational signature of the protein upon ligand binding has been monitored by Nuclear Inelastic Scattering (NIS), also called Nuclear Resonant Vibrational Spectroscopy (NRVS). The NIS data thus obtained have also been calculated by quantum mechanical (QM) density functional theory (DFT) coupled with molecular mechanics (MM) methods. The calculations presented here show that the heme ruffling in NP2 is a consequence of the interaction with the protein matrix. Structure optimizations of the heme and its ligands with DFT retain the characteristic saddling and ruffling only if the protein matrix is taken into account. Furthermore, simulations of the NIS data by QM/MM calculations suggest that the pH dependence of the binding of NO, but not of CN(-) and histamine, might be a consequence of the protonation state of the heme carboxyls.

Tyrosine triad at the interface between the Rieske iron-sulfur protein, cytochrome c1 and cytochrome c2 in the bc1 complex of Rhodobacter capsulatus.

A triad of tyrosine residues (Y152-154) in the cytochrome c(1) subunit (C1) of the Rhodobacter capsulatus cytochrome bc(1) complex (BC1) is ideally positioned to interact with cytochrome c(2) (C2). Mutational analysis of these three tyrosines showed that, of the three, Y154 is the most important, since its mutation to alanine resulted in significantly reduced levels, destabilization, and inactivation of BC1. A second-site revertant of this mutant that regained photosynthetic capacity was found to have acquired two further mutations-A181T and A200V. The Y152Q mutation did not change the spectral or electrochemical properties of C1, and showed wild-type enzymatic C2 reduction rates, indicating that this mutation did not introduce major structural changes in C1 nor affect overall activity. Mutations Y153Q and Y153A, on the other hand, clearly affect the redox properties of C1 (e.g. by lowering the midpoint potential as much as 117 mV in Y153Q) and the activity by 90% and 50%, respectively. A more conservative Y153F mutant on the other hand, behaves similarly to wild-type. This underscores the importance of an aromatic residue at position Y153, presumably to maintain close packing with P184, which modeling indicates is likely to stabilize the sixth heme ligand conformation.

Intramolecular electron transfer in sulfite-oxidizing enzymes: probing the role of aromatic amino acids.

Sulfite oxidase (SO) is a molybdoheme enzyme that is important in sulfur catabolism, and mutations in the active site region are known to cause SO deficiency disorder in humans. This investigation probes the effects that mutating aromatic residues (Y273, W338, and H337) in the molybdenum-containing domain of human SO have on both the intramolecular electron transfer (IET) rate between the molybdenum and iron centers using laser flash photolysis and on catalytic turnover via steady-state kinetic analysis. The W338 and H337 mutants show large decreases in their IET rate constants (k (ET)) relative to the wild-type values, suggesting the importance of these residues for rapid IET. In contrast, these mutants are catalytically competent and exhibit higher k (cat) values than their corresponding k (ET), implying that these two processes involve different conformational states of the protein. Redox potential investigations using spectroelectrochemistry revealed that these aromatic residues close to the molybdenum center affect the potential of the presumably distant heme center in the resting state (as shown by the crystal structure of chicken SO), suggesting that the heme may be interacting with these residues during IET and/or catalytic turnover. These combined results suggest that in solution human SO may adopt different conformations for IET and for catalysis in the presence of the substrate. For IET the H337/W338 surface residues may serve as an alternative-docking site for the heme domain. The similarities between the mutant and wild-type EPR spectra indicate that the active site geometry around the Mo(V) center is not changed by the mutations studied here.

NMR studies of nitrophorin distal pocket side chain effects on the heme orientation and seating of NP2 as compared to NP1.

The nitrophorins (NP) of the adult blood-sucking insect Rhodnius prolixus fall into two pairs based on sequence identity (NP1,4 (90%) and NP2,3 (79%)), which differ significantly in the size of side chains of residues which contact the heme. These residues include those in the distal pocket of NP2 (I120) and NP1 (T121) and the "belt" that surrounds the heme of NP2 (S40, F42), and NP1(A42, L44). To determine the importance of these residues and others conserved or very similar for the two pairs, including L122(123), L132(133), appropriate mutants of NP2 and NP1 have been prepared and studied by (1)H NMR spectroscopy. Wild-type NP2 has heme orientation ratio (A:B) of 1:8 at equilibrium, while wild-type NP1 has A:B ~1:1 at equilibrium. Another difference between NP2 and NP1 is in the heme seating with regard to His57(59). It is found that among the distal pocket residues investigated, the residue most responsible for heme orientation and seating is I120(T121). F42(L44) and L106(F107) may also be important, but must be investigated in greater detail.

Oxidation and loss of heme in soluble guanylyl cyclase from Manduca sexta.

Oxidation and loss of heme in soluble guanylyl/guanylate cyclase (sGC), the nitric oxide receptor, is thought to be a major contributor to cardiovascular disease and is the target of compounds BAY 58-2667 and HMR1766. Using spectroelectrochemical titration, we found a truncated sGC to be highly stable in the ferrous state (234 mV) and to bind ferrous heme tightly even in the presence of NO, despite the NO-induced release of the proximal histidine. In contrast, oxidized sGC readily loses ferric heme to myoglobin (0.47 ± 0.02 h(-1)). Peroxynitrite, the presumed cellular oxidant, readily oxidizes sGC in 5 mM glutathione.

Unprecedented peroxidase-like activity of Rhodnius prolixus nitrophorin 2: identification of the [FeIV=O Por•]+ and [FeIV=O Por](Tyr38•) intermediates and their role(s) in substrate oxidation.

We have identified a novel enzymatic reaction for nitrophorin 2 (NP2), a heme protein previously characterized as a nitric oxide carrier in the saliva of the Rhodnius prolixus insect. NP2 exhibited levels of peroxidase activity comparable to those of the bifunctional peroxidases (KatGs), despite their heme pocket structural differences (heme ruffling, Tyr38 and Tyr85 in hydrogen bonding interactions with the propionates in NP2). The intermediates of the peroxidase-like reaction of NP2 were identified by Electron Paramagnetic Resonance (EPR) and electronic absorption spectroscopies. The EPR spectrum consistent with an [Fe(IV)=O Por•]+ species was detected at pH <7. At pH ≥ 7, the change from a strong to a weak antiferromagnetic coupling interaction for the [Fe(IV)=O Por•]+ species was accompanied by the subsequent formation of an [Fe(IV)=O Por](Tyr•) intermediate. Tyr38 was shown to be the unique naturally occurring radical site in NP2. The Y38F mutant stabilized the radical on the tyrosine in hydrogen-bonding interaction with the other heme propionate (Tyr85). Kinetic studies using stopped-flow electronic absorption spectrophotometry revealed that the [Fe(IV)=O Por•]+ species reacts with histamine and norepinephrine in a peroxidase-like manner. Our findings demonstrate that NP2 has pH-dependent dual function: at the acidic pH of the insect saliva the protein behaves as a NO carrier, and, if exposed to the higher pH of the tissues and capillaries of the host, NP2 is able to bind histamine or it can efficiently inactivate norepinephrine through a peroxidase-like reaction, in the presence of hydrogen peroxide. Accordingly, the unprecedented peroxidase-like activity of NP2 is concluded to be a key biological function.

Effects of interdomain tether length and flexibility on the kinetics of intramolecular electron transfer in human sulfite oxidase.

Sulfite oxidase (SO) is a vitally important molybdenum enzyme that catalyzes the oxidation of toxic sulfite to sulfate. The proposed catalytic mechanism of vertebrate SO involves two intramolecular one-electron transfer (IET) steps from the molybdenum cofactor to the iron of the integral b-type heme and two intermolecular one-electron steps to exogenous cytochrome c. In the crystal structure of chicken SO [Kisker, C., et al. (1997) Cell 91, 973-983], which is highly homologous to human SO (HSO), the heme iron and molybdenum centers are separated by 32 A and the domains containing these centers are linked by a flexible polypeptide tether. Conformational changes that bring these two centers into greater proximity have been proposed [Feng, C., et al. (2003) Biochemistry 42, 5816-5821] to explain the relatively rapid IET kinetics, which are much faster than those theoretically predicted from the crystal structure. To explore the proposed role(s) of the tether in facilitating this conformational change, we altered both its length and flexibility in HSO by site-specific mutagenesis, and the reactivities of the resulting variants have been studied using laser flash photolysis and steady-state kinetics assays. Increasing the flexibility of the tether by mutating several conserved proline residues to alanines did not produce a discernible systematic trend in the kinetic parameters, although mutation of one residue (P105) to alanine produced a 3-fold decrease in the IET rate constant. Deletions of nonconserved amino acids in the 14-residue tether, thereby shortening its length, resulted in more drastically reduced IET rate constants. Thus, the deletion of five amino acid residues decreased IET by 70-fold, so that it was rate-limiting in the overall reaction. The steady-state kinetic parameters were also significantly affected by these mutations, with the P111A mutation causing a 5-fold increase in the sulfite K(m) value, perhaps reflecting a decrease in the ability to bind sulfite. The electron paramagnetic resonance spectra of these proline to alanine and deletion mutants are identical to those of wild-type HSO, indicating no significant change in the Mo active site geometry.

Scanning chimeragenesis: the approach used to change the substrate selectivity of fatty acid monooxygenase CYP102A1 to that of terpene omega-hydroxylase CYP4C7.

CYP102A1 is a highly active, water-soluble, bacterial monooxygenase enzyme that contains both substrate-binding heme and diflavin reductase subunits, both in a single polypeptide. Recently we developed a procedure which uses the known structure of the substrate-bound heme domain of CYP102A1 and its sequence homology with a cytochrome P450 of unknown structure, both of which react with a common substrate but produce different products, to create recombinant enzymes which have substrate selectivity different from that of CYP102A1, and produce the product of the enzyme of unknown structure. Insect CYP4C7, a terpene hydroxylase from the cockroach, was chosen as the cytochrome P450 of unknown structure, and farnesol was chosen as the substrate. CYP102A1 oxidizes farnesol to three products (2,3-epoxyfarnesol, 10,11-epoxyfarnesol, and 9-hydroxyfarnesol), whereas CYP4C7 produces 12-hydroxyfarnesol as the major product. In earlier work it was found that the chimera C(78-82,F87L) showed a change in substrate selectivity from fatty acids to farnesol, and was approximately sixfold more active than wild-type CYP102A1 (Chen et al. in J Biol Inorg Chem 13:813-824, 2008), but neither it nor any other earlier chimera produced 12-hydroxyfarnesol. In this work we added amino acid residues 327-332, to create six new full-length, functional chimeric proteins. Four of these, the most active of which was C(78-82,F87L,328-330), produce 12-hydroxyfarnesol as the major product, with approximately twofold increase in turnover number as compared with wild-type CYP102A1 toward farnesol. Methylfarnesoate was metabolized to 12-hydroxymethylfarnesoate (70%) and 10,11-epoxymethylfarnesoate (juvenile hormone III) (30%). The latter is metabolized to 65% 12-hydroxy-10,11-epoxymethylfarnesoate and 35% 15-hydroxy-10,11-epoxymethylfarnesoate. Substitution of residues 328-330, APA, by VPL was crucial to accomplishing this change in product.

1H and 13C NMR spectroscopic studies of the ferriheme resonances of three low-spin complexes of wild-type nitrophorin 2 and nitrophorin 2(V24E) as a function of pH.

The ferriheme resonances of the low-spin (S = 1/2) complexes of wild-type (wt) nitrophorin 2 (NP2) and its heme pocket mutant NP2(V24E) with imidazole (ImH), histamine (Hm), and cyanide (CN(-)) as the sixth ligand have been investigated by NMR spectroscopy as a function of pH (4.0-7.5). For the three wt NP2 complexes, the ratio of the two possible heme orientational isomers, A and B, remains almost unchanged (ratio of A:B approximately 1:6 to 1:5) over this wide pH range. However, strong chemical exchange cross peaks appear in the nuclear Overhauser effect spectroscopy/exchange spectroscopy (NOESY/EXSY) spectra for the heme methyl resonances at low pH (pH* 4.0-5.5), which indicate chemical exchange between two species. We have shown these to be two different exogenous ImH or Hm orientations that are denoted B and B', with the ImH plane nearly parallel and perpendicular to the ImH plane of the protein-provided His57, respectively. The wt NP2-CN complex also shows EXSY cross peaks due to chemical exchange, which is shown to be a result of interchange between two ruffling distortions of the heme. The same ruffling distortion interchange is also responsible for the ImH and Hm chemical exchange. For the three NP2(V24E) ligand complexes, no EXSY cross peaks are observed, but the A:B ratios change dramatically with pH. The fact that heme favors the A orientation highly for NP2(V24E) at low pH as compared with wt NP2 is believed to be due to the steric effect of the V24E mutation. The existence of the B' species at lower pH for wt NP2 complexes and the increase in A heme orientation at lower pH for NP2(V24E) are believed to be a result of a change in structure near Glu53 when it is protonated at low pH. 1H{13C} heteronuclear multiple quantum coherence (HMQC) spectra are very helpful for the assignment of heme and nearby protein side chain resonances.