Kinetic studies of iron deposition in horse spleen ferritin using H2O2 and O2 as oxidants

The reaction of horse spleen ferritin (HoSF) with Fe2+ at pH 6.5 and 7.5 using O2, H2O2 and 1:1 a mixture of both showed that the iron deposition reaction using H2O2 is approximately 20- to 50-fold faster than the reaction with O2 alone. When H2O2 was added during the iron deposition reaction initiated with O2 as oxidant, Fe2+ was preferentially oxidized by H2O2, consistent with the above kinetic measurements. Both the O2 and H2O2 reactions were well defined from 15 to 40 degrees C from which activation parameters were determined. The iron deposition reaction was also studied using O2 as oxidant in the presence and absence of catalase using both stopped-flow and pumped-flow measurements. The presence of catalase decreased the rate of iron deposition by approximately 1.5-fold, and gave slightly smaller absorbance changes than in its absence. From the rate constants for the O2 (0.044 s(-1)) and H2O2 (0.67 s(-1)) iron-deposition reactions at pH 7.5, simulations of steady-state H2O2 concentrations were computed to be 0.45 microM. This low value and reported Fe2+/O2 values of 2.0-2.5 are consistent with H2O2 rapidly reacting by an alternate but unidentified pathway involving a system component such as the protein shell or the mineral core as previously postulated [Biochemistry 22 (1983) 876; Biochemistry 40 (2001) 10832].     

Kinetics of O2 evolution from H2O2 catalyzed by the oxygen-evolving complex: investigation of the S1-dependent reaction

The evolution of O2 from H2O2 catalyzed by the oxygen-evolving complex (OEC) in darkness was examined with photosystem II reaction center complex preparations from spinach. Flash illumination of dark-adapted reaction centers was used to make S0-enriched or S1-enriched complexes. The membranes catalyzed O2 evolution from H2O2 when preset to either the S0 or S1 state. However, only the S0-state reaction was inhibited by carbonyl cyanide m-chlorophenylhydrazone and dependent on chloride. These results indicate that (1) the S0-dependent and S1-dependent catalytic cycles can be separated by flash illumination, (2) the S0-dependent reaction involves the formation of the S2 state, and (3) the S1-dependent reaction does not involve the formation of the S2 or S3 states. A kinetic study of the S1-dependent reaction revealed a rapid equilibrium ordered mechanism in which (1) the binding of Ca(II) must precede the binding of H2O2 to the OEC and (2) the reaction of Ca(II) with the free enzyme is at thermodynamic equilibrium such that Ca(II) does not necessarily dissociate after each catalytic cycle.     

Effect of Hb-encapsulation with vesicles on H2O2 reaction and lipid peroxidation

We encapsulated a purified and concentrated hemoglobin (Hb) solution with a phospholipid bilayer membrane to form Hb vesicles (particle diameter, ca. 250 nm) for the development of artificial oxygen carriers. Reaction of Hb inside the vesicle with hydrogen peroxide (H(2)O(2)) is one of the important safety issues to be clarified and compared with a free Hb solution. During the reaction of the Hb solution with H(2)O(2), metHb (Fe(III)) and ferrylHb (Fe(IV)=O) are produced, and H(2)O(2) is decomposed by the catalase-like reaction of Hb. The aggregation of discolored Hb products due to heme degradation is accompanied by the release of iron (ferric ion). On the other hand, the concentrated Hb within the Hb vesicle reacts with H(2)O(2) that permeated through the bilayer membrane, and the same products as the Hb solution are formed inside the vesicle. However, there is no turbidity change, no particle diameter change of the Hb vesicles, and no peroxidation of lipids comprising the vesicles after the reaction with H(2)O(2). Furthermore, no free iron is detected outside the vesicle, though ferric ion is released from the denatured Hb inside the vesicle, indicating the barrier effect of the bilayer membrane against the permeation of ferric ion. When vesicles composed of egg york lecithin (EYL) as unsaturated lipids are added to the mixture of Hb and H(2)O(2), the lipid peroxidation is caused by ferrylHb and hydroxyl radical generated from reaction of the ferric iron with H(2)O(2), whereas no lipid peroxidation is observed in the case of the Hb vesicle dispersion because the saturated lipid membrane of the Hb vesicle should prevent the interaction of the ferrylHb or ferric iron with the EYL.     

Reactions of peroxyl radicals with Fe(H(2)O)(6)(2+)

The reactions of RO(2)* radicals with Fe(H(2)O)(6)(2+) were studied, R[double bond]H; CH(3); CH(2)COOH; CH(2)CN; CH(2)C(CH(3))(2)OH; CH(2)OH; CHCl(2)/CCl(3). All these processes involve the following reactions: Fe(H(2)O)(6)(2+)+RO(2)*<==>(H(2)O)(5)Fe(III)[bond]OOR(2+) K(1) approximately 250 M(-1); (H(2)O)(5)Fe(III)[bond]OOR(2+)+H(3)O(+)/H(2)O-->Fe(H(2)O)(6)(3+)+ROOH+H(2)O/OH(-); (H(2)O)(5)Fe(III)[bond]OOR(2+)+2Fe(H(2)O)(6)(2+)-->3Fe(H(2)O)(6)(3+)+ROH; 2 RO(2)*-->Products; RO(2)*+(H(2)O)(5)Fe(III)[bond]OOR(2+)-->Fe(H(2)O)(6)(2+)+products. The values of k(1) and k(3) [reaction is clearly not an elementary reaction] approach the ligand exchange rate of Fe(H(2)O)(6)(2+), i.e. these reactions follow an inner sphere mechanism and the rate determining step is the ligand exchange step. The rate of reaction is several orders of magnitude faster than that of the Fenton reaction. Surprisingly enough the K(1) values are nearly independent of the redox potential of the radical and are considerably higher than calculated from the relevant redox potentials. These results indicate that the ROO(-) ligands considerably stabilise the Fe(III) complex, this stabilisation is smaller for radicals with electron withdrawing groups which raise the redox potential of the radical but decrease the basicity of the ROO(-) ligands, two effects which seem to nearly cancel each other. Finally, the results clearly indicate that reaction (5) is relatively fast and affects the nature of the final products. The contribution of these reactions to oxidation processes involving 'Fenton-like' processes is discussed.     

Photosystem II energy coupling in chloroplasts with H2O2 as electron donor.

NH2OH-treated, non-water-splitting chloroplasts can oxidize H2O2 to O2 through Photosystem II at substantial rates (100--250 muequiv . h-1 . mg-1 chlorophyll with 5 mM H2O2) using 2,5-dimethyl-p-benzoquinone as an electron acceptor in the presence of the plastoquinone antagonist dibromothymoquinone. This H2O2 leads to Photosystem II leads to dimethylquinone reaction supports phosphorylation with a P/e2 ratio of 0.25--0.35 and proton uptake with H+/e values of 0.67 (pH 8)--0.85 (pH 6). These are close to the P/e2 value of 0.3--0.38 and the H+/e values of 0.7--0.93 found in parallel experiments for the H2O leads to Photosystem II leads to dimethylquinone reaction in untreated chloroplasts. Semi-quantitative data are also presented which show that the donor leads to Photosystem II leads to dibromothymoquinone (leads to O2) reaction can support phosphorylation when the donor used is a proton-releasing reductant (benzidine, catechol) but not when it is a non-proton carrier (I-, ferrocyanide).     

Hydrogen peroxide formation during iron deposition in horse spleen ferritin using O2 as an oxidant

The reaction of Fe2+ with O2 in the presence of horse spleen ferritin (HoSF) results in deposition of FeOH3 into the hollow interior of HoSF. This reaction was examined at low Fe2+/HoSF ratios (5-100) under saturating air at pH 6.5-8.0 to determine if H2O2 is a product of the iron deposition reaction. Three methods specific for H2O2 detection were used to assess H2O2 formation: (1) a fluorometric method with emission at 590 nm, (2) an optical absorbance method based on the reaction H2O2 + 3I- + 2H+ = I3- + 2H2O monitored at 340 nm for I3- formation, and (3) a differential pulsed electrochemical method that measures O2 and H2O2 concentrations simultaneously. Detection limits of 0.25, 2.5, and 5.0 microM H2O2 were determined for the three methods, respectively. Under constant air-saturation conditions (20% O2) and for a 5-100 Fe2+/HoSF ratio, Fe2+ was oxidized and the resulting Fe3+ was deposited within HoSF but no H2O2 was detected as predicted by the reaction 2Fe2+ + O2 + 6H2O = 2Fe(OH)3 + H2O2 + 4H+. Two other sets of conditions were also examined: one with excess but nonsaturating O2 and another with limiting O2. No H2O2 was detected in either case. The absence of H2O2 formation under these same conditions was confirmed by microcoulometric measurements. Taken together, the results show that under low iron loading conditions (5-100 Fe2+/HoSF ratio), H2O2 is not produced during iron deposition into HoSF using O2 as an oxidant. This conclusion is inconsistent with previous, carefully conducted stoichiometric and kinetic measurements [Xu, B., and Chasteen, N. D. (1991) J. Biol. Chem. 266, 19965], predicting that H2O2 is a quantitative product of the iron deposition reaction with O2 as an oxidant, even though it was not directly detected. Possible explanations for these conflicting results are considered.     

水分胁迫下苹果叶片的H_2O_2代谢

轻度水分胁迫下苹果叶片Pr迅速升高 ,CAT活性变化不大 ,NaHSO3 处理能显著降低叶内H2 O2 含量 ,表明光呼吸的加强促进了H2 O2 产生可能是叶内H2 O2大量积累的主要原因 ;中度水分胁迫下叶片AsA含量的下降和Mehler反应的增强都非常明显 ,DDTC和AsA处理都能有效降低叶内H2 O2 积累 ,但MV处理的作用不大 ,说明叶片H2 O2 主要来源于Mehler反应 ,AsA降解造成叶片对H2 O2 清除能力的下降是其积累的根本原因 ;严重水分胁迫时 ,NaHSO3 和DDTC都不能有效地减轻叶内H2 O2 积累 ,光呼吸和Mehler反应都可能不是H2 O2 的主要来源     

Scavenging of H2O2 and production of oxygen by horseradish peroxidase

Peroxidases catalyze many reactions, the most common being the utilization of H2O2 to oxidize numerous substrates (peroxidative mode). Peroxidases have also been proposed to produce H2O2 via utilization of NAD(P)H, thus providing oxidant either for the first step of lignification or for the "oxidative burst" associated with plant-pathogen interactions. The current study with horseradish peroxidase characterizes a third type of peroxidase activity that mimics the action of catalase; molecular oxygen is produced at the expense of H2O2 in the absence of other reactants. The oxygen production and H2O2-scavenging activities had temperature coefficients, Q10, of nearly 3 and 2, which is consistent with enzymatic reactions. Both activities were inhibited by autoclaving the enzyme and both activities had fairly broad pH optima in the neutral-to-alkaline region. The apparent Km values for the oxygen production and H2O2-scavenging reactions were near 1.0 mM H2O2. Irreversible inactivation of horseradish peroxidase by exposure to high concentrations of H2O2 coincided with the formation of an absorbance peak at 670 nm. Addition of superoxide dismutase (SOD) to reaction mixtures accelerated the reaction, suggesting that superoxide intermediates were involved. It appears that horseradish peroxidase is capable of using H2O2 both as an oxidant and as a reductant. A model is proposed and the relevance of the mechanism in plant-bacterial systems is discussed.     

Dimethylthiourea consumption reflects H2O2 concentrations and severity of acute lung injury

Even though dimethylthiourea (DMTU) effectively scavenges O2 metabolites in vitro, it is often unclear if scavenging of O2 metabolites is the mechanism by which DMTU decreases tissue injury in biological models. Since DMTU not only scavenges O2 metabolites but is also consumed in a dose-response manner following reaction with hydrogen peroxide (H2O2) in vitro, we wondered whether DMTU would also be consumed by O2 metabolites in biological systems and if DMTU consumption would then reflect O2 metabolite concentrations and O2 metabolite-mediated injury. Our results supported this possibility. We found that selected nonprotecting concentrations of DMTU were consumed in isolated rat lungs perfused with H2O2 and that the amounts of DMTU consumed reflected both the added amounts of H2O2 and the corresponding degrees of H2O2-induced acute edematous injury. DMTU consumption was relatively specific for reaction with H2O2 occurring in isolated lungs that were injured by H2O2 but not lungs injured by elastase, oleic acid, histamine, or a venous pressure challenge. Our results suggest that measurement of DMTU consumption may be useful for assessing the presence and toxicity of O2 metabolites and the specificity of the protective effects of DMTU in biological systems.     

Co-oxidation of salicylate and cholesterol during the oxidation of metmyoglobin by H2O2

The reaction between metmyoglobin and H2O2 proceeds with oxidation of the hemo-protein iron to a higher valence state and consumption of the peroxide. This reaction is further associated with (a) O2 evolution; (b) hydroxylation of the aromatic compound salicylate to yield a set of dihydroxybenzoic acid derivatives (analyzed by HPLC with electrochemical detection); (c) autoxidation of cholesterol with formation of 3 beta-hydroxy-5-alpha-cholest-6-ene-5-hydroperoxide; and (d) formation of electronically excited states detected by low-level chemiluminescence. The heterolytic scission of the O-O bond of hydroperoxides by metmyoglobin causes the formation of an oxidizing equivalent capable of promoting peroxidation of linoleate and arachidonate (as indicated by the parallel formation of thiobarbituric acid-reactive material and an enhancement of chemiluminescence intensity). The identity of the oxidizing equivalent(s) is discussed in terms of the formation of a relatively stable higher state of oxidation of heme Fe (FeIV-OH or FeV = O) as well as on possible intermediate species derived during the decomposition of H2O2 by metmyoglobin, such as HO.and 1O2. These species might be involved either simultaneously or sequentially in the peroxidation of fatty acids as well as in the tissue damage associated with the formation of H2O2 in ischemic-reperfusion states.     

Gamma-radiolysis of N2O-saturated formate solutions. A chain reaction

In the radiolysis of aqueous formate-containing solutions a chain reaction (i, ii) proceeds in the presence of N2O. CO2-. + N2O + H2O----CO2 + N2 + .OH + OH- (i) .OH + HCO2-.----CO2-. + H2O (ii) The chain length depends on the dose rate and the N2O concentration but not on the formate concentration. Typically, G(CO2) approximately 140 molecules (100 eV)-1 is found, with an equivalent amount of N2, at a dose rate of 3 X 10(-3) Gy s-1. The rate constant for the rate-determining step in this chain reaction has been calculated at k(i) = 1600 dm3 mol-1 s-1. The possible relevance of this chain reaction in radiation biological studies is briefly discussed.     

Determination of H2O2-dependent generation of singlet oxygen from human saliva with a novel chemiluminescence probe

Singlet oxygen (1O2) has been shown to play an important role in salivary defense system, but its generation process and level from human saliva remain uncertain due to the lack of a reliable detection method. We have previously reported 4,4'(5')-bis[2-(9-anthryloxy)ethylthio]tetrathiafulvalene (BAET) as a novel chemiluminescence probe for 1O2. In this work, the probe is successfully used to characterize H2O2-dependent generation of 1O2 from saliva in real time. However, the yield of 1O2 is found to be very low, for example, being about 0.13 nmol from 200 microL saliva in the presence of 1 mM of hydrogen peroxide over a 5-s reaction period. The result is also compared with that obtained with another 1O2 probe 2-methyl-6-phenyl-3,7-dihydroimidazo[1,2-a]pyrazin-3-one (CLA), demonstrating that, besides 1O2, the other reactive oxygen species such as hydroxyl radical may also be involved in the reaction of saliva with H2O2. Furthermore, the present study shows that the selectivity of BAET for 1O2 is much higher than that of CLA and thus BAET is highly suited for the detection of 1O2 in the presence of other reactive oxygen species in biological systems.     

Kinetic studies of iron deposition catalyzed by recombinant human liver heavy and light ferritins and Azotobacter vinelandii bacterioferritin using O2 and H2O2 as oxidants

The discrepancy between predicted and measured H(2)O(2) formation during iron deposition with recombinant heavy human liver ferritin (rHF) was attributed to reaction with the iron protein complex [Biochemistry 40 (2001) 10832-10838]. This proposal was examined by stopped-flow kinetic studies and analysis for H(2)O(2) production using (1) rHF, and Azotobacter vinelandii bacterial ferritin (AvBF), each containing 24 identical subunits with ferroxidase centers; (2) site-altered rHF mutants with functional and dysfunctional ferroxidase centers; and (3) recombinant human liver light ferritin (rLF), containing no ferroxidase center. For rHF, nearly identical pseudo-first-order rate constants of 0.18 s(-1) at pH 7.5 were measured for Fe(2+) oxidation by both O(2) and H(2)O(2), but for rLF, the rate with O(2) was 200-fold slower than that for H(2)O(2) (k = 0.22 s(-1)). A Fe(2+)/O(2) stoichiometry near 2.4 was measured for rHF and its site altered forms, suggesting formation of H(2)O(2). Direct measurements revealed no H(2)O(2) free in solution 0.5-10 min after all Fe(2+) was oxidized at pH 6.5 or 7.5. These results are consistent with initial H(2)O(2) formation, which rapidly reacts in a secondary reaction with unidentified solution components. Using measured rate constants for rHF, simulations showed that steady-state H(2)O(2) concentrations peaked at 14 muM at approximately 600 ms and decreased to zero at 10-30 s. rLF did not produce measurable H(2)O(2) but apparently conducted the secondary reaction with H(2)O(2). Fe(2+)/O(2) values of 4.0 were measured for AvBF. Stopped-flow measurements with AvBF showed that both H(2)O(2) and O(2) react at the same rate (k = 0.34 s(-1)), that is faster than the reactions with rHF. Simulations suggest that AvBF reduces O(2) directly to H(2)O without intermediate H(2)O(2) formation.     

Synergistic damage from H2O2 and OH radicals in irradiated cells

The anoxic sensitization of bacterial spores by added H2O2 has been studied. Two mechanistic pathways for damage from H2O2 were found; one of these requires the presence of OH radicals. For this kind of damage, the relationship between H2O2 and OH appears to be that they are reactants. O-2 (and/or HO2), the product of such a reaction, is likely the agent which actually causes damage. These results with reagent H2O2 are compared with results of experiments in which H2O2 and OH are present as radiolytic products.     

Significance of catalase in peroxisomal fatty acyl-CoA beta-oxidation: NADH oxidation by acetoacetyl-CoA and H2O2

To clarify the significance of catalase in peroxisomes, we have examined the effect of aminotriazole treatment of rats on the activity of beta-hydroxybutyryl-CoA dehydrogenase in liver peroxisomes. When the effect of H2O2 on the dehydrogenase activity was examined using an extract of liver peroxisomes from aminotriazole-treated rats, the acetoacetyl-CoA-dependent oxidation of NADH was found to increase considerably on the addition of dilute H2O2. Such an effect of H2O2 was not seen on the beta-hydroxybutyryl-CoA-dependent reduction of NAD nor with extracts from untreated animals. We then noticed that similar NADH oxidation was caused non-enzymatically by a mixture of acetoacetyl-CoA and H2O2. The oxidation was dependent on both acetoacetyl-CoA and H2O2, and was blocked by scavengers of oxyradicals such as ascorbate and ethanol. Degradation products formed during the reaction of acetoacetyl-CoA with H2O2 had no NADH oxidizing activity, indicating that effective oxidant(s) were generated during the reaction of H2O2 with acetoacetyl-CoA. No other fatty acyl-CoA so far examined nor acetoacetate could replace acetoacetyl-CoA in this reaction. Therefore, if H2O2 were to be accumulated in peroxisomes, it would decrease both NADH and acetoacetyl-CoA, thus affecting the fatty acyl-CoA beta-oxidation system. These results, together with our previous finding that peroxisomal thiolase was significantly inactivated by H2O2 [Hashimoto, F. & Hayashi, H. (1987) Biochim. Biophys. Acta 921, 142-150] suggest that the role of catalase in peroxisomes is at least in part to protect the fatty acyl-CoA beta-oxidation system from the deleterious action of H2O2.     

A functional model for pMMO (particulate methane monooxygenase): Hydroxylation of alkanes with H2O2 catalyzed by beta-diketiminatocopper(II) complexes

The reaction of copper(II) complexes supported by a series of beta-diketiminate ligands ((R1,R2)L, [(Dipp)N-C(R(2))-C(R(1))-C(R(2))-N(Dipp)](-), Dipp=2,6-diisopropylphenyl; see ) and H(2)O(2) has been examined spectroscopically at a low temperature. The beta-diketiminatocopper(II) complexes with R(2)=H (no substituent on the beta-carbon) provided a copper-oxygen intermediate that exhibited the same spectroscopic features as those of the bis(mu-oxo)dicopper(III) complex generated by the reaction of corresponding beta-diketiminatocopper(I) complex and O(2). On the other hand, the beta-diketiminatocopper(II) complexes with methyl substituent on the beta-carbon (R(2)=Me) did not produce such an intermediate in the same reaction. The beta-diketiminatocopper(II) complexes carrying an electron-withdrawing substituent on the alpha-carbon (R(1)=NO(2) or CN) but no beta-substituent (R(2)=H) exhibited a high catalytic activity in the oxygenation reaction of alkanes with H(2)O(2). Mechanism of the catalytic oxygenation reaction as well as the substituent effects of the ligands on the copper(II)-H(2)O(2) reactivity is discussed.     

Supply of O2 regulates O2 demand during utilization of reserves of poly-beta-hydroxybutyrate in N2-fixing soybean bacteroids.

A liquid reaction medium containing dissolved air and oxyleghaemoglobin, but no energy-yielding substrate, was supplied to bacteroids confined in a stirred flow reaction chamber. The relative oxygenation of the leghaemoglobin in the chamber was determined automatically by spectrophotometry of the effluent solution, and the concentrations of free, dissolved O2 ([O2]) and rates of O2 consumption were calculated. Dissolved CO2 and NH3 from N2 fixation were determined in fractions of the effluent solution. Bacteroids utilized endogenous reserves of poly-beta-hydroxybutyrate (PHB), which were depleted by 9.2% during a typical 5 h-long experiment. Stepwise increases in flow rate (increasing supply of O2) initially produced a drop in O2 demand and resulted in a rise in [O2] and a decline in N2 fixation. Subsequently, O2 demand rose (presumably because of increased mobilization of substrate from PHB) and [O2] declined to a low level. N2 fixation was fully restored, or even enhanced, within 15-20 min of establishment of a new, steady [O2]. This pattern of regulation by O2 supply was completely eliminated by adding low concentrations (20-50 microM) of oxidizable substrate (succinate, malate, ethanol) to the reaction medium. During endogenous activity, rates of CO2 evolution were proportional to, but less than, rates of O2 consumption up to 5.4 nmol O2 min-1 mg-1, above which CO2 evolution exceeded O2 consumption. These and other features of endogenous activity are discussed in relation to sustaining N2 fixation by nodules in vivo.     

H/2H isotope effect in redox reactions of cytochrome c.

The rate of reaction of ferro- and ferricytochrome c (C(II) and C(III) with ferri- and ferrocyanide and of C(III) with 02- and CO2- was determined in H2O and in 2H2O in the temperature range 5-35 degrees C. No isotope effect was evident in any of the reductions of C(III); the apparent energy of activation was identical in H2O and 2H2O. An isotope effect with kH2O/k2H2O = 1.25 to 1.85, depending on pH for instance was observed in the oxidation of C(II), in the slow phase of oxidation which involves conformational changes. An interpretation (supported by evidence from previous work) involving water molecules in the close vicinity of the reaction site on the protein is discussed.     

Gentamicin nucleotidyltransferase. Stereochemical inversion at phosphorus in enzymatic 2'-deoxyadenylyl transfer to tobramycin

Gentamicin nucleotidyltransferase-catalyzed reaction of (Sp)-[alpha-17O]dATP with tobramycin produced 2"-(2'-deoxyadenosine 5'-[17O]phosphoryl)tobramycin. The configuration at phosphorus in this product was shown to be Rp by chemical degradation to chiral [17O, 18O]dAMP using a stereochemically defined procedure, and determination of the configuration at phosphorus in this product. Periodate-base treatment of 2"-(2'-deoxyadenosine 5'-[17O]phosphoryl)tobramycin followed by NaBH4 reduction produced (2-glyceryl)-[17O]dAMP, which upon snake venom phosphodiesterase-catalyzed hydrolysis in H(2)18O produced [17O,18O] dAMP. The configuration at phosphorus in this product was shown to be S by enzymatic phosphorylation to [17O,18O]dATP, adenylylcyclase (Bordetella pertussis)-catalyzed cyclization to 3',5'-cyclic [17O,18O]dAMP, and 31P NMR analysis of the ethyl esters. Since snake venom phosphodiesterase-catalyzed hydrolyses proceed with retention of configuration at phosphorus, (Sp)-[17O,18O]dAMP must have been produced from (Rp)-(2-glyceryl)-[17O]dAMP; and since the chemical degradation to the latter compound did not involve cleavage of any bonds to phosphorus, the initial enzymatic product must have been (Rp)-2"-(2'-deoxyadenosine 5'-[17O]phosphoryl)tobramycin. Therefore, nucleotidyl transfer catalyzed by gentamicin nucleotidyl-transferase proceeds with inversion of configuration at phosphorus, and the reaction mechanism involves an uneven number of phosphotransfer steps. Inasmuch as this is an uncomplicated two-substrate group transfer reaction, the mechanism probably involves direct nucleotidyl transfer from the nucleoside triphosphate to the aminoglycoside. The B. pertussis adenylylcyclase reaction was shown to proceed with inversion at phosphorus, as has been established for other adenylylcyclases.