Allosteric behavior of platelet myosin.

The allosteric properties of platelet actomyosin and myosin have been further studied. At pH 7.2, both exhibit sigmoid kinetics with at least two interacting ATP binding sites. At pH 8.9, the velocity versus substrate curve is shifted to the right and becomes more sigmoidal. In contrast, at pH 5.5, the enzyme appears to follow hyperbolic kinetics and the K_m is reduced. In the presence of 1.4 m urea, the sigmoidicity is lost and the enzyme obeys Michaelis-Menten kinetics. The effect of ADP on the ATPase activity was also investigated. ADP shows characteristics of a competitive inhibitor; it increases K_m (shifts sigmoid curve to the right) without affecting V. When the enzyme is desensitized by low pH (5.5) or urea (1.4 m), the allosteric interaction is abolished without impairing the catalytic activity and ADP is no longer inhibitory. These findings suggest that platelet myosin possesses two interacting sites and that ADP binds to the allosteric site which appears to be different from the catalytic site.     

Activation and Inhibition of Histone Deacetylase 8 by Monovalent Cations

The metal-dependent histone deacetylases (HDACs) catalyze hydrolysis of acetyl groups from acetyllysine side chains and are targets of cancer therapeutics. Two bound monovalent cations (MVCs) of unknown function have been previously observed in crystal structures of HDAC8; site 1 is near the active site, whereas site 2 is located >20 Å from the catalytic metal ion. Here we demonstrate that one bound MVC activates catalytic activity (K_1/2 = 3.4 mm for K⁺), whereas the second, weaker-binding MVC (K_1/2 = 26 mm for K⁺) decreases catalytic activity by 11-fold. The weaker binding MVC also enhances the affinity of the HDAC inhibitor suberoylanilide hydroxamic acid by 5-fold. The site 1 MVC is coordinated by the side chain of Asp-176 that also forms a hydrogen bond with His-142, one of two histidines important for catalytic activity. The D176A and H142A mutants each increase the K_1/2 for potassium inhibition by ≥40-fold, demonstrating that the inhibitory cation binds to site 1. Furthermore, the MVC inhibition is mediated by His-142, suggesting that this residue is protonated for maximal HDAC8 activity. Therefore, His-142 functions either as an electrostatic catalyst or a general acid. The activating MVC binds in the distal site and causes a time-dependent increase in activity, suggesting that the site 2 MVC stabilizes an active conformation of the enzyme. Sodium binds more weakly to both sites and activates HDAC8 to a lesser extent than potassium. Therefore, it is likely that potassium is the predominant MVC bound to HDAC8 in vivo.     

The use of 8-azido-ATP and 8-azido-ADP as photoaffinity labels of the ATP synthase in submitochondrial particles: Evidence for a mechanism of ATP hydrolysis involving two independent catalytic sites?

8-Azido-ATP is a substrate for the ATP synthase in submitochondrial particles with a V_max equal to 6% of the V_max with ATP. The K_m values for 8-azido-ATP are similar to those for ATP. ATP synthase in submitochondrial particles can bind maximally 2 mol 8-N-ATP or 8-N-ADP per mole and the inhibition of ATP hydrolysis by covalently bound N-ATP or N-ADP is proportional to the saturation of the enzyme with inhibitor, similar to the results obtained with isolated F₁. Both 8-N-ATP and 8-N-ADP are bound mainly to the β subunits and at all levels of saturation the distribution of the label is 77% to the β and 23% to the α subunits. It is proposed that the binding of 8-azido-AXP itself is mainly to the β subunit, but that part of the nitreno radicals formed during excitation with light reacts with an amino acid of the α subunit, due to the location of the binding site at an interface between a β and an α subunit. Partial saturation with 8-N-ATP, under conditions that the concentration of 8-azido-ATP during the incubation is intermediate between the low and high K_m values, does not abolish the apparent negative cooperativity of ATP hydrolysis. It is concluded that this apparent cooperativity is not due to the presence of two different catalytic sites, nor to a cooperativity between the two catalytic sites, but to interaction between the catalytic sites and regulatory sites.     

Origin of apparent negative cooperativity of F1-ATPase

In order to get insight into the origin of apparent negative cooperativity observed for F₁-ATPase, we compared ATPase activity and ATPMg binding of mutant subcomplexes of thermophilic F₁-ATPase, α_(W463F)3β_(Y341W)3γ and α_{(K175A/T176A/W463F)3}β_(Y341W)3γ. For α_(W463F)3β_(Y341W)3γ, apparent K_m's of ATPase kinetics (4.0 and 233 μM) did not agree with apparent K_m's deduced from fluorescence quenching of the introduced tryptophan residue (on the order of nM, 0.016 and 13 μM). On the other hand, in case of α_{(K175A/T176A/W463F)3}β_(Y341W)3γ, which lacks noncatalytic nucleotide binding sites, the apparent K_m of ATPase activity (10 μM) roughly agreed with the highest K_m of fluorescence measurements (27 μM). The results indicate that in case of α_(W463F)3β_(Y341W)3γ, the activating effect of ATP binding to noncatalytic sites dominates overall ATPase kinetics and the highest apparent K_m of ATPase activity does not represent the ATP binding to a catalytic site. In case of α_{(K175A/T176A/W463F)3}β_(Y341W)3γ, the K_m of ATPase activity reflects the ATP binding to a catalytic site due to the lack of noncatalytic sites. The Eadie-Hofstee plot of ATPase reaction by α_{(K175A/T176A/W463F)3}β_(Y341W)3γ was rather linear compared with that of α_(W463F)3β_(Y341W)3γ, if not perfectly straight, indicating that the apparent negative cooperativity observed for wild-type F₁-ATPase is due to the ATP binding to catalytic sites and noncatalytic sites. Thus, the frequently observed K_m's of 100-300 μM and 1-30 μM range for wild-type F₁-ATPase correspond to ATP binding to a noncatalytic site and catalytic site, respectively.     

Kinetic studies on the (Na+ + K+)-dependent ATPase. Evidence for coexisting sites for Na+, K+ and Mg2+

Na⁺-ATPase activity of a dog kidney (Na⁺ + K⁺)-ATPase enzyme preparation was inhibited by a high concentration of NaCl (100 mM) in the presence of 30 μM ATP and 50 μM MgCl₂, but stimulated by 100 mM NaCl in the presence of 30 μM ATP and 3 mM MgCl₂. The $\text{K}{}_{0.5}$ for the effect of MgCl₂ was near 0.5 mM. Treatment of the enzyme with the organic mercurial thimerosal had little effect on Na⁺-ATPase activity with 10 mM NaCl but lessened inhibition by 100 mM NaCl in the presence of 50 μM MgCl₂. Similar thimerosal treatment reduced (Na⁺ + K⁺)-ATPase activity by half but did not appreciably affect the $\text{K}{}_{0.5}$ for activation by either Na⁺ or K⁺, although it reduced inhibition by high Na⁺ concentrations. These data are interpreted in terms of two classes of extracellularly-available low-affinity sites for Na⁺: Na⁺-discharge sites at which Na⁺-binding can drive E₂-P back to E₁-P, thereby inhibiting Na⁺-ATPase activity, and sites activating E₂-P hydrolysis and thereby stimulating Na⁺-ATPase activity, corresponding to the K⁺-acceptance sites. Since these two classes of sites cannot be identical, the data favor co-existing Na⁺-discharge and K⁺-acceptance sites. Mg²⁺ may stimulate Na⁺-ATPase activity by favoring E₂-P over E₁-P, through occupying intracellular sites distinct from the phosphorylation site or Na⁺-acceptance sites, perhaps at a coexisting low-affinity substrate site. Among other effects, thimerosal treatment appears to stimulate the Na⁺-ATPase reaction and lessen Na⁺-inhibition of the (Na⁺ + K⁺)-ATPase reaction by increasing the efficacy of Na⁺ in activating E₂-P hydrolysis.     

The role of effector molecules in regulating ribulosebisphosphate carboxylase activity

Ribulosebisphosphate carboxylase can exist in two forms having different kinetic properties. The fraction of enzyme present in each of the two forms is determined by both the absolute and the relative amounts of substrates and other effector molecules in solution with the enzyme. High CO₂ levels induce formation of an active CO₂ form of the enzyme while high ribulosebisphosphate (RuBP) levels cause formation of a much less active form. The CO₂ form is characterized by a high K_m(RuBP), low K_m(CO2), and relatively high V. The ribulosebisphosphate form has comparatively lower K_m(RuBP) and V and higher K_m(CO2). CO₂ appears to bind before RuBP in the reaction sequence catalyzed by the CO₂ form; this catalytic binding order is apparently reversed in the RuBP form. Steady state rates of enzyme reaction reflect the contributions of both these forms. A brief model, based on the cooperative effects of binding at a small number of catalytic or activator sites in the multimeric enzyme, is presented to account for the changes in enzyme activity with varying substrate and effector molecule concentrations.     

Steady-state kinetics of ADP-arsenate and ATP-synthesis in Rhodospirillum rubrum chromatophores

(1) Rates of ATP synthesis and ADP-arsenate synthesis catalyzed by Rhodospirillum rubrum chromatophores were determined with the firefly luciferase method and by a coupled enzyme assay involving hexokinase and glucose-6-phosphate dehydrogenase. (2) V_m for ADP-arsenate synthesis was about 2-times lower than V_m for ATP-synthesis. With saturating [ADP], K(As_i) was about 20% higher than K(P_i). With saturating [anion], K(ADP) was during arsenylation about 20% lower than during phosphorylation. (3) Plots of $\text{1}\text{v}$ vs. $\text{1}\text{[}\text{substrate}\text{]}$ were non-linear at low concentrations of the fixed substrate. The non-linearity was such as to suggest a positive cooperativity between sites binding the variable substrate, resulting in an increased $\text{V}{}_{\mathrm{<mtext>m</mtext>}}\text{K}{}_{\mathrm{<mtext>m</mtext>}}$ ratio. High concentrations of the fixed substrate cause a similar increase in $\text{V}{}_{\mathrm{<mtext>m</mtext>}}\text{K}{}_{\mathrm{<mtext>m</mtext>}}$, but abolish the cooperativity of the sites binding the variable substrate. (4) Low concentrations of inorganic arsenate (As_i) stimulate ATP synthesis supported by low concentrations of P_i and ADP about 2-fold. (5) At high ADP concentrations, the apparent K_i of As_i for inhibition of ATP-synthesis was 2–3-times higher than the apparent K_m of As_i for arsenylation; the apparent K_i of P_i for inhibition of ADP-arsenate synthesis was about 40% lower than the apparent K_m of P_i for ATP synthesis. (6) The results are discussed in terms of a model in which P_i and As_i compete for binding to a catalytic as well as an allosteric site. The interaction between these sites is modulated by the ADP concentration. At high ADP concentrations, interaction between these sites occurs only when they are occupied with different species of anion.     

Role of chloride ion as an allosteric activator of angiotensin-converting enzyme

The nature of chloride ion as an activator of angiotensin-converting enzyme was studied by a series of kinetic experiments with hog plasma enzyme preparation. The enzyme required the presence of chloride ion for its full catalytic activity, but its requirement of monovalent anion was not absolute. The K_A value for the enzymechloride binding was estimated to be about 150 mm in all cases regardless of the peptide substrates employed. In the presence of chloride ion, the activity of the enzyme was increased, but its optimum pH was shifted gradually to the alkaline region up to pH 8.2 depending on the concentration of chloride ion. In addition, in the presence of chloride ion, the apparent K_m values were reduced markedly while the V_max values were not much altered; for example, for the hydrolysis of angiotensin I decapeptide, the K_m value decreased by a factor of 50 while only an 18% increase in V_max was observed when the enzyme was saturated with chloride ion. The result suggests that chloride ion acts as a conformational modifier inducing the affinity of synergistic binding of substrate.     

Origin of the pKa shift of the catalytic lysine in acetoacetate decarboxylase

The pK_a value of Lys115, the catalytic residue in acetoacetate decarboxylate, was calculated using atomic coordinates of the X-ray crystal structure with consideration of the protonation states of all titratable sites in the protein. The calculated pK_a value of Lys115 (pK_a(Lys115)) was unusually low (≈6) in agreement with the experimentally measured value. Although charged residues impact pK_a(Lys115) considerably in the native protein, the significant pK_a(Lys115) downshift in the protein with respect to aqueous solution was mainly due to loss of the solvation energy in the catalytic active site relative to bulk water.     

Studies on the reaction mechanism of DT diaphorase. Action of dead-end inhibitors and effects of phospholipids.

The relationship between the inhibition of DT diaphorase [NAD(P)H dehydrogenase (quinone): EC.1.6.99.2] by 4-hydroxycoumarin and the 1,3-indandione derivates was investigated. Evidence is presented that these two classes of anticoagulants, although both acting as competitive inhibitors with respect to NAD(P)H, bind to different sites of the enzyme in a synergistic fashion. These findings are interpreted as indicative of a cooperativity between different substrate-binding sites of the enzyme.Neutral phospholipids exert effects on partially purified rat-liver DT diaphorase similar to those earlier obtained with nonionic detergents. The effects concern several kinetic parameters of the enzyme, including V, K_m for electron donor and acceptor, and K_i for various inhibitors. The changes in the kinetic parameters vary in extent and direction according to the individual phospholipids.     

PH dependence of the alpha-glucose 1,6-diphosphate inhibition of hexokinase II.

α-Glucose 1,6-diphosphate is a much better inhibitor of hexokinase II than 1,5-anhydroglucitol 6-phosphate or glucose 6-phosphate (Glc-6-P) at pH 6–7 and poorer at higher pH. Because the K_i of Glc-6-P is pH independent, the observed pH effects are attributed to the phosphate group at C-1 which is bound as a monoanion to a specific site but which is excluded as a dianion. None of the following kinetic properties of the hexokinase II reaction varies greatly with pH: V, K_m of glucose and K_m of ATP.     

Activation and inactivation of rabbit liver pyridoxamine (pyridoxine) 5'-phosphate oxidase activity by urea and other solutes.

Urea, guanidine hydrochloride, and neutral salts both activate and denature pyridoxamine (pyridoxine) 5′-phosphate oxidase (EC 1.4.3.5) from rabbit liver. Activation occurs at lower concentrations (e.g. 2-2.5 m for urea) of these compounds and is rapid and reversible. Greater structural changes leading to inactivation occur slowly under “activating conditions” but rapidly at higher concentrations of urea. Both reversibly and irreversibly inactivated species are formed. Activation by urea does not involve either dissociation of the enzyme to subunits or aggregation to multimers, and there is little disruption of protein secondary structure. The V and K_m for substrates, K_i for product, and the rate of release of product from the enzyme are increased by urea, and substrate inhibition is decreased; urea has little effect on the reactivity of reduced enzyme with oxygen. Both flavin and tryptophanyl fluorescence increase in the presence of urea; at lower concentrations of urea (≤2 m), there is a rapid increase followed by slower, sigmoidal increases. The polarization of flavin fluorescence of the oxidase is increased upon the addition of 2 m urea, which corresponds to the initial enhancement of protein and flavin fluorescence intensities, and then decreases. The near-ultraviolet-visible absorption spectra of native enzyme and that treated with 2 m urea are only slightly different; however, a considerable change at the flavin-binding site is reflected by the circular dichroism spectra. Hence, it appears that urea yields a rapidly formed, “activated” species of the oxidase that is changed primarily at the active site in a manner that allows increased dissociation of substrate and product.     

pH Effects on the Activity and Regulation of the NAD Malic Enzyme

The NAD malic enzyme shows a pH optimum of 6.7 when complexed to Mg²⁺ and NAD⁺ but shifts to 7.0 when the catalytically competent enzyme-substrate (E-S) complex forms upon binding malate⁻². This is characteristic of an induced conformational change. The slope of the V_max or V_max/K_m profiles is steeper on the alkaline side of the pH optimum. The K_m for malate increases markedly under alkaline conditions but is not greatly affected by pH values below the optimum. The loss of catalysis on the acidic side is due to protonation of a single residue, pK 5.9, most likely histidine. Photooxidation inactivation with methylene blue showed that a histidine is required for catalytic activity. The location of this residue at or near the active site is revealed by the protection against inactivation offered by malate. Three residues, excluding basic residues such as lysine (which have also been shown to be vital for catalytic activity, must be appropriately ionized for malate decarboxylation to proceed optimally. Two of these residues directly participate in the binding of substrates and are essential for the decarboxylation of malate. A pK of 7.6 was determined for the two residues required by the E-S complex to achieve an active state, this composite value representing both histidine and cysteine suggests that both have decisive roles in the operation of the enzyme. A major change in the enzyme takes place as protonation nears the pH optimum, this is recorded as a change in the enzyme's intrinsic affinity for malate (K_{m pH6.7} = 9.2 millimolar, K_{m pH7.7} = 28.3 millimolar). Similar changes in K_m have been observed for the NAD malic enzyme as it shifts from dimer to tetramer. It is most likely that the third ionizable group (probably a cysteine) revealed by the V_max/K_m profile is needed for optimal activity and is involved in the association-dissociation behavior of the enzyme.     

The uptake and metabolism of urea by Chara australis: IV. Symport with sodium—A slip model for the high and low affinity systems

We have previously investigated the electrogenic influx of urea in Chara, and the urea- and sodium-dependent membrane current. We have shown that there is a sodium-stimulated component of urea influx and a urea-stimulated component of sodium influx, and that these are of the same size. We conclude that the electrogenic inward transport of urea, and of its analogues acetamide and acrylamide, is by sodium symport, with a stoichiometric ratio of 1∶1. The kinetics of the fluxes and currents show two different K _M values for sodium in different cells and two different kinds of kinetics for the effect of urea on membrane current, one of which fits the Michaelis-Menten equation, while the other shows a maximum and fits the difference of two Michaelis-Menten terms, suggesting a phenomenon like cis-inhibition. Similarities in kinetic characteristics between the inhibitory site and the electrically silent uptake site (System II) lead us to suggest that the same protein may be responsible for both the low-K _M, electrogenic influx of urea (System I) and the high-K _M, electrically silent influx by System II. We suggest a “slip” model for urea uptake in Chara.     

Interaction between gamma-carboxyglutamic acid and glutamate dehydrogenase

The amino acid γ-carboxyglutamic acid, recently discovered in some vitamin K-dependent blood-clotting factors, shows interesting kinetic effects on glutamate dehydrogenase. It is not metabolized by the enzyme; it is a powerful competitive inhibitor (K_i = 3.8 × 10^?4 m) with respect to NAD⁺ and glutamate. On the other hand the reverse reaction is activated by γ-carboxyglutamate, both K_m and V being altered; this effect is additive with the well-known activating effect of ADP.     

Kinetic properties of aspartate transport in rat heart mitochondrial inner membranes.

The mitochondrial glutamate-aspartate exchange carrier catalyzes the electrogenic exchange of intramitochondrial aspartate for extramitochondrial glutamate. Protons are cotransported with glutamate in a 1:1 ratio. In the present study, the effects of pH and glutamate concentration on glutamate entry into intact mitochondria were determined. Hydrogen ions were found to decrease the K_m for glutamate entry. In addition, using glutamate-loaded submitochondrial particles, aspartate transport into the particles was measured as a function of internal and external glutamate concentrations, pH, and electrical potential across the membrane. Glutamate, was a competitive inhibitor of aspartate transport when both amino acids were present on the same side of the membrane, while H⁺ was a noncompetitive inhibitor of aspartate entry into the particles. A decrease in glutamate concentration on the inside of the particles brought about a parallel decrease in V and K_m for aspartate outside of the particles, thus suggesting a ping-pong mechanism for the carrier. The uncoupling agent, carbonyl cyanide p-trifluoromethoxy-phenylhydrazone (FCCP), lowered both the K_m and V of aspartate transport, while the effect on V was somewhat larger. Data obtained in the presence of KSCN was similar to that obtained with FCCP, and therefore it is concluded that both K_m and V changes are dependent on a change of electrical potential across the membrane. A model for the carrier is proposed, which is consistent with the data presented. The model includes a single binding site specific for either glutamate or aspartate, and a separate binding site for the cotransported proton. The affinity of the binding site for protons is increased by simultaneous glutamate binding, but decreased by aspartate binding. The data suggest that an increase in the membrane potential increases the mobility of the charged carrier-aspartate complex, but also facilitates some additional step in the exchange cycle involving subsequent return of the carrier to the matrix side of the membrane. The additional membrane-potential-dependent step could be proton binding on the cytosolic side of the carrier.     

Activation and inhibition of mitochondrial adenosine triphosphatase by various anions and other agents

The activating or inhibiting actions of a variety of anion species and of oligomycin, aurovertin and Dio-9 on the ATPase of a sonic particle preparation of rat liver mitochondria have been characterized by measurements of the relevantV _max,K _i andK _m values.The normalV _max was increased by a factor near 7 by the anions: dichromate, chromate, pyrophosphate, orthophosphate, orthoarsenate and sulphate. The fully activating concentration varied from about 2 mM for dichromate to 150 mM for sulphate. The increase inV _max was accompanied by a time-dependent decrease in (K _i)ADP, but there was no change in (K _m)ATP. The increase inV _max by the activating anions was abolished by aurovertin; but in presence of oligomycin, the lowV _max was increased by the activating anions by the same factor as theV _max in absence of oligomycin.Certain anions, notably azide, decreasedV _max, but did not affect (K _i)ADP or (K _m)ATP. The decrease inV _max by azide and oligomycin were approximately additive. Even at high concentration, Dio-9 was without detectable effect on the ATPase, but it had a gramicidinlike effect on the intact mitochondria.The specificity of the ATPase for ATP relative to GTP was found to be attributable to the high value of (V _max)ATP compared with (V _max)GTP. The values of (K _m)ATP and (K _m)GTP were virtually the same.Some rationalization of these and other supporting observations is attempted in terms of present knowledge of the constitution of the ATPase complex.     

Pleiotropy Can Be Effectively Estimated Without Counting Phenotypes Through the Rank of a Genotype–Phenotype Map

Although pleiotropy, the capability of a gene to affect multiple phenotypes, has been well known as one of the common gene properties, a quantitative estimation remains a great challenge, simply because of the phenotype complexity. Not surprisingly, it is hard for general readers to understand how, without counting phenotypes, gene pleiotropy can be effectively estimated from the genetics data. In this article we extensively discuss the Gu-2007 method that estimated pleiotropy from the protein sequence analysis. We show that this method is actually to estimate the rank (K) of genotype–phenotype mapping that can be concisely written as K = min(r, P_min), where P_min is the minimum pleiotropy among all legitimate measures including the fitness components, and r is the rank of mutational effects of an amino acid site. Together, the effective gene pleiotropy (K_e) estimated by the Gu-2007 method has the following meanings: (i) K_e is an estimate of K = min(r, P_min), the rank of a genotype–phenotype map; (ii) K_e is an estimate for the minimum pleiotropy P_min only if P_min < r; (iii) the Gu-2007 method attempted to estimate the pleiotropy of amino acid sites, a conserved proxy to the true gene pleiotropy; (iv) with a sufficiently large phylogeny such that the rank of mutational effects at an amino acid site is r → 19, one can estimate P_min between 1 and 19; and (v) K_e is a conserved estimate of K because those slightly affected components in fitness have been effectively removed by the estimation procedure. In addition, we conclude that mutational pleiotropy (number of traits affected by a single mutation) cannot be estimated without knowing the phenotypes.     

Molecular mechanism of poly(ADP-ribosyl)ation by PARP1 and identification of lysine residues as ADP-ribose acceptor sites

Poly(ADP-ribose) polymerase 1 (PARP1) synthesizes poly(ADP-ribose) (PAR) using nicotinamide adenine dinucleotide (NAD) as a substrate. Despite intensive research on the cellular functions of PARP1, the molecular mechanism of PAR formation has not been comprehensively understood. In this study, we elucidate the molecular mechanisms of poly(ADP-ribosyl)ation and identify PAR acceptor sites. Generation of different chimera proteins revealed that the amino-terminal domains of PARP1, PARP2 and PARP3 cooperate tightly with their corresponding catalytic domains. The DNA-dependent interaction between the amino-terminal DNA-binding domain and the catalytic domain of PARP1 increased V_max and decreased the K_m for NAD. Furthermore, we show that glutamic acid residues in the auto-modification domain of PARP1 are not required for PAR formation. Instead, we identify individual lysine residues as acceptor sites for ADP-ribosylation. Together, our findings provide novel mechanistic insights into PAR synthesis with significant relevance for the different biological functions of PARP family members.     

Contrasting catalytic profiles of multiheme nitrite reductases containing CxxCK heme-binding motifs

The multiheme cytochromes from Thioalkalivibrio nitratireducens (TvNiR) and Escherichia coli (EcNrfA) reduce nitrite to ammonium. Both enzymes contain His/His-ligated hemes to deliver electrons to their active sites, where a Lys-ligated heme has a distal pocket containing a catalytic triad of His, Tyr, and Arg residues. Protein-film electrochemistry reveals significant differences in the catalytic properties of these enzymes. TvNiR, but not EcNrfA, requires reductive activation. Spectroelectrochemistry implicates reduction of His/His-ligated heme(s) as being key to this process, which restricts the rate of hydroxide binding to the ferric form of the active-site heme. The K _M describing nitrite reduction by EcNrfA varies with pH in a sigmoidal manner that is consistent with its modulation by (de)protonation of a residue with pK _a ≈ 7.6. This residue is proposed to be the catalytic His in the distal pocket. By contrast, the K _M for nitrite reduction by TvNiR decreases approximately linearly with increase of pH such that different features of the mechanism define this parameter for TvNiR. In other regards the catalytic properties of TvNiR and EcNrfA are similar, namely, the pH dependence of V _max and the nitrite dependence of the catalytic current–potential profiles resolved by cyclic voltammetry, such that the determinants of these properties appear to be conserved.