Individual heme a and heme a3 contributions to the Soret absorption spectrum of the reduced bovine cytochrome c oxidase.

Bovine cytochrome c oxidase (CcO) contains two hemes, a and a3, chemically identical but differing in coordination and spin state. The Soret absorption band of reduced aa3-type cytochrome c oxidase consists of overlapping bands of the hemes a2+ and a32+. It shows a peak at ∼444 nm and a distinct shoulder at ∼425 nm. However, attribution of individual spectral lineshapes to hemes a2+ and a32+ in the Soret is controversial. In the present work, we characterized spectral contributions of hemes a2+ and a32+ using two approaches. First, we reconstructed bovine CcO heme a2+ spectrum using a selective Ca2+-induced spectral shift of the heme a2+. Second, we investigated photobleaching of the reduced Thermus thermophilus ba3- and bovine aa3-oxidases in the Soret induced by femtosecond laser pulses in the Q-band. The resolved spectra show splitting of the electronic B0x-, B0y-transitions of both reduced hemes. The heme a2+ spectrum is shifted to the red relative to heme a32+ spectrum. The ∼425 nm shoulder is mostly attributed to heme a32+.

Mechanism of Inhibition of Cytochrome c Oxidase by Triton X-100.

It is known that Triton X-100 (TX) reversibly inhibits activity of cytochrome c oxidase (CcO). The mechanism of inhibition is analyzed in this work. The action of TX is not directed to the reaction of CcO with cytochrome c, does not cause transition of the enzyme to the "slow" form, and is not associated with monomerization of the enzyme complex. TX completely suppresses oxygen reduction by CcO, but inhibition is prevented and partially reversed by dodecyl-ß-D-maltoside (DDM), a detergent used to maintain CcO in solution. A 1/1 stoichiometry competition is shown between DDM and TX for binding to CcO, with Ki = 0.3 mM and affinity of DDM for the enzyme of 1.2 mM. TX interaction with the oxidized enzyme induces spectral response with maximum at 421 nm and [TX]1/2 = 0.28 mM, presumably associated with heme a3. When CcO interacts with excess of H2O2 TX affects equilibrium of the oxygen intermediates of the catalytic center accelerating the FI-607 → FII-580 transition, inhibits generation of O2·- by the enzyme, and, to a lesser extent, suppresses the catalase partial activity. The observed effects can be explained by inhibition of the conversion of the intermediate FII-580 to the free oxidized state during the catalytic cycle. TX suppresses intraprotein electron transfer between hemes a and a3 during enzyme turnover. Partial peroxidase activity of CcO remains relatively resistant to TX under conditions that block oxidase reaction effectively. These features indicate an impairment of the K proton channel conductivity. We suggest that TX interacts with CcO at the Bile Acid Binding Site (BABS) that is located on the subunit I at the K-channel mouth and contacts with amphipathic regulators of CcO [Buhrow et al. (2013) Biochemistry, 52, 6995-7006]. Apparently, TX mimics the physiological ligand of BABS, whereas the DDM molecule mimics an endogenous phospholipid bound at the edge of BABS that controls effective affinity for the ligand.

Interaction of Cytochrome C Oxidase with Steroid Hormones.

Estradiol, testosterone and other steroid hormones inhibit cytochrome c oxidase (CcO) purified from bovine heart. The inhibition is strongly dependent on concentration of dodecyl-maltoside (DM) in the assay. The plots of Ki vs [DM] are linear for both estradiol and testosterone which may indicate an 1:1 stoichiometry competition between the hormones and the detergent. Binding of estradiol, but not of testosterone, brings about spectral shift of the oxidized CcO consistent with an effect on heme a33+. We presume that the hormones bind to CcO at the bile acid binding site described by Ferguson-Miller and collaborators. Estradiol is shown to inhibit intraprotein electron transfer between hemes a and a3. Notably, neither estradiol nor testosterone suppresses the peroxidase activity of CcO. Such a specific mode of action indicates that inhibition of CcO activity by the hormones is associated with impairing proton transfer via the K-proton channel.

Effect of Ca2+ on the redox potential of heme a in cytochrome c oxidase.

Subunit I of cytochrome c oxidase (CcO) from mitochondria and many bacteria contains a cation binding site (CBS) located at the outer positively charged aqueous phase not far from heme a. Binding of Ca2+ with the CBS in bovine CcO inhibits activity of the enzyme 2-3 -fold [Vygodina, T., Kirichenko, A. & Konstantinov A.A. (2013) Direct Regulation of Cytochrome c Oxidase by Calcium Ions, PLoS One.8 e74436]. Here we show that binding of Ca2+ at CBS of bovine CcO shifts Em of heme a to the positive by 15-20 mV. Na+ ions that bind to the same site and compete with Ca2+ do not affect Em of heme a and also prevent and reverse the effect of Ca2+. No effect of Ca2+ or EGTA is observed on Em of heme a with the wild type bacterial oxidases from R.sphaeroides or P.denitrificans that contain tightly-bound calcium at the site. In the D477A mutant CcO from P. denitrificans that binds Ca2+ reversibly like the mitochondrial CcO, calcium shifts redox titration curve of heme a to the positive by ∼35-50 mV that is in good agreement with the results of electrostatic calculations; however, as shown earlier, it does not inhibit CcO activity of the mutant enzyme. Therefore the data do not support the proposal that the inhibitory effect of Ca2+ on CcO activity may be explained by the Ca2+-induced shift of Em of heme a. Rather, Ca2+ retards electron transfer by inhibition of charge dislocation in the exit part of the proton channel H in mammalian CcO, that is absent in the bacterial oxidases.

Cytochrome c oxidase inhibition by calcium at physiological ionic composition of the medium: Implications for physiological significance of the effect.

Cytochrome c oxidase (CcO) from mammalian mitochondria binds Ca2+ and Na+ in a special cation binding site. Binding of Ca2+ brings about partial inhibition of the enzyme while Na+ competes with Ca2+ for the binding site and protects the enzyme from the inhibition [Vygodina, T., Kirichenko, A. and Konstantinov, A.A. (2013). Direct Regulation of Cytochrome c oxidase by Calcium Ions. PLoS One 8(9): e74436]. In the original studies, the inhibition was found to depend significantly on the ionic composition of the buffer. Here we describe inhibition of CcO by Ca2+ in media containing the main ionic components of cytoplasm (150mM KCl, 12mM NaCl and 1mM MgCl2). Under these conditions, Ca2+ inhibits CcO with effective Ki of 20-26µM, that is an order of magnitude higher than determined earlier in the absence of Na+. At physiological value of ionic strength, the inhibition can be observed at any turnover number of CcO, rather than only at low TN (<10s-1) as found previously. The inhibition requires partially oxidized state of cytochrome c and is favored by high ionic strength with a sharp transition at 0.1-0.2M. The high Ki=20-26µM found for CcO inhibition by calcium matches closely the known value of "Km" for Ca2+-induced activation of the mitochondrial calcium uniporter. The inhibition of CcO by Ca2+ is proposed to modulate mitochondrial Ca2+-uptake via the mitochondrial calcium uniporter, promote permeability transition pore opening and induce reduction of Mia40 in the mitochondrial intermembrane space.

Calcium ions inhibit reduction of heme a in bovine cytochrome c oxidase.

The effect of Ca(2+) on the rate of heme a reduction by dithionite and hexaammineruthenium (RuAm) was studied in the cyanide-complexed bovine cytochrome oxidase (CcO). The rate of heme a reduction is proportional to RuAm concentration below 300 µM with kv of 0.53×10(6) M(-1) s(-1). Ca(2+) inhibits the rate of heme a reduction by dithionite by ∼25%. As the reaction speeds up with increased concentrations of RuAm, the inhibition by Ca(2+) disappears. The inhibition of heme a reduction may contribute to recently described partial inhibition of CcO by Ca(2+) in the enzymatic assays. The inhibitory effect of Ca(2+) on heme a reduction indicates that ET through heme a may be coupled to proton movement in the exit part of the proton channel H.

Cation binding site of cytochrome c oxidase: progress report.

Cytochrome c oxidase from bovine heart binds Ca(2+) reversibly at a specific Cation Binding Site located near the outer face of the mitochondrial membrane. Ca(2+) shifts the absorption spectrum of heme a, which allowed earlier the determination of the kinetic and equilibrium characteristics of the binding, and, as shown recently, the binding of calcium to the site inhibits cytochrome oxidase activity at low turnover rates of the enzyme [Vygodina, Т., Kirichenko, A., Konstantinov, A.A (2013). Direct Regulation of Cytochrome c Oxidase by Calcium Ions. PloS ONE 8, e74436]. This paper summarizes further progress in the studies of the Cation Binding Site in this group presenting the results to be reported at 18th EBEC Meeting in Lisbon, 2014. The paper revises specificity of the bovine oxidase Cation Binding Site for different cations, describes dependence of the Ca(2+)-induced inhibition on turnover rate of the enzyme and reports very high affinity binding of calcium with the "slow" form of cytochrome oxidase. This article is part of a Special Issue entitled: 18th European Bioenergetic Conference. Guest Editors: Manuela Pereira and Miguel Teixeira.

Direct regulation of cytochrome c oxidase by calcium ions.

Cytochrome c oxidase from bovine heart binds Ca(2+) reversibly at a specific Cation Binding Site located near the outer face of the mitochondrial membrane. Ca(2+) shifts the absorption spectrum of heme a, which allowed previously to determine the kinetics and equilibrium characteristics of the binding. However, no effect of Ca(2+) on the functional characteristics of cytochrome oxidase was revealed earlier. Here we report that Ca(2+) inhibits cytochrome oxidase activity of isolated bovine heart enzyme by 50-60% with Ki of ∼1 µM, close to Kd of calcium binding with the oxidase determined spectrophotometrically. The inhibition is observed only at low, but physiologically relevant, turnover rates of the enzyme (∼10 s(-1) or less). No inhibitory effect of Ca(2+) is observed under conventional conditions of cytochrome c oxidase activity assays (turnover number >100 s(-1) at pH 8), which may explain why the effect was not noticed earlier. The inhibition is specific for Ca(2+) and is reversed by EGTA. Na(+) ions that compete with Ca(2+) for binding with the Cation Binding Site, do not affect significantly activity of the enzyme but counteract the inhibitory effect of Ca(2+). The Ca(2+)-induced inhibition of cytochrome c oxidase is observed also with the uncoupled mitochondria from several rat tissues. At the same time, calcium ions do not inhibit activity of the homologous bacterial cytochrome oxidases. Possible mechanisms of the inhibition are discussed as well as potential physiological role of Ca(2+) binding with cytochrome oxidase. Ca(2+)- binding at the Cation Binding Site is proposed to inhibit proton-transfer through the exit part of the proton conducting pathway H in the mammalian oxidases.

Cytochrome c oxidase: charge translocation coupled to single-electron partial steps of the catalytic cycle.

The paper presents a survey of time-resolved studies of charge translocation by cytochrome c oxidase coupled to transfer of the 1st, 2nd 3rd and 4th electrons in the catalytic cycle. Single-electron photoreduction experiments carried out with the A-class cytochrome c oxidases of aa(3) type from mitochondria, Rhodobacter sphaeroides and Paracoccus denitrificans as well as with the ba(3)-type oxidase from Thermus thermophilus indicate that the protonmotive mechanisms, although similar, may not be identical for different partial steps in the same enzyme species, as well as for the same single-electron transition in different oxidases. The pattern of charge translocation coupled to transfer of a single electron in the A-class oxidases confirms major predictions of the original model of proton pumping by cytochrome oxidase [Artzatbanov, V. Y., Konstantinov, A. A. and Skulachev, V.P. "Involvement of Intramitochondrial Protons in Redox Reactions of Cytochrome a." FEBS Lett. 87: 180-185]. The intermediates and partial electrogenic steps observed in the single-electron photoreduction experiments may be very different from those observed during oxidation of the fully reduced oxidase by O(2) in the "flow-flash" studies. .

Cytochrome c oxidase: Intermediates of the catalytic cycle and their energy-coupled interconversion.

Several issues relevant to the current studies of cytochrome c oxidase catalytic mechanism are discussed. The following points are raised. (1) The terminology currently used to describe the catalytic cycle of cytochrome oxidase is outdated and rather confusing. Presumably, it would be revised so as to share nomenclature of the intermediates with other oxygen-reactive heme enzymes like P450 or peroxidases. (2) A "catalytic cycle" of cytochrome oxidase involving complete reduction of the enzyme by 4 electrons followed by oxidation by O(2) is a chimera composed artificially from two partial reactions, reductive and oxidative phases, that never operate together as a true multi-turnover catalytic cycle. The 4e(-) reduction-oxidation cycle would not serve a paradigm for oxygen reduction mechanism and protonmotive function of cytochrome oxidase. (3) The foremost role of the K-proton channel in the catalytic cycle may consist in securing faultless delivery of protons for heterolytic O-O bond cleavage in the oxygen-reducing site, minimizing the danger of homolytic scission reaction route. (4) Protonmotive mechanism of cytochrome oxidase may vary notably for the different single-electron steps in the catalytic cycle.

Circular dichroism spectra of cytochrome c oxidase.

Circular dichroism spectra of bovine heart aa(3)-type cytochrome c oxidase have been studied with a major focus on the Soret band π → π* transitions, B(0(x,y)), in the two iron porphyrin groups of the enzyme. The spectra of the fully reduced and fully oxidized enzyme as well as of its carbon monoxide and cyanide complexes have been explored. In addition, CD spectra of the reduced and oxidized ba(3)-type cytochrome c oxidase from Thermus thermophilus were recorded for comparison. An attempt is made to interpret the CD spectra of cytochrome c oxidase with the aid of a classical model of dipole-dipole coupled oscillators taking advantage of the known 3D crystal structure of the enzyme. Simultaneous modeling of the CD and absorption spectra shows that in the bovine oxidase, the dipole-dipole interactions between the hemes a and a(3), although contributing significantly, cannot account either for the lineshape or the magnitude of the experimental spectra. However, adding the interactions of the hemes with 22 aromatic amino acid residues located within 12 Å from either of the two heme groups can be used to model the CD curves for the fully reduced and fully oxidized oxidase with reasonable accuracy. Interaction of the hemes with the peptide bond transition dipoles is found to be insignificant. The modeling indicates that the CD spectra of cytochrome oxidase in both the reduced and oxidized states are influenced significantly by interaction with Tyr244 in the oxygen-reducing center of the enzyme. Hence, CD spectroscopy may provide a useful tool for monitoring the redox/ionization state of this residue. The modeling confirms wide energy splitting of the orthogonal B(x) and B(y) transitions in the porphyrin ring of heme a.

Partial steps of charge translocation in the nonpumping N139L mutant of Rhodobacter sphaeroides cytochrome c oxidase with a blocked D-channel.

The N139L substitution in the D-channel of cytochrome oxidase from Rhodobacter sphaeroides results in an approximately 15-fold decrease in the turnover number and a loss of proton pumping. Time-resolved absorption and electrometric assays of the F --> O transition in the N139L mutant oxidase result in three major findings. (1) Oxidation of the reduced enzyme by O(2) shows approximately 200-fold inhibition of the F --> O step (k approximately 2 s(-1) at pH 8) which is not compatible with enzyme turnover ( approximately 30 s(-1)). Presumably, an abnormal intermediate F(deprotonated) is formed under these conditions, one proton-deficient relative to a normal F state. In contrast, the F --> O transition in N139L oxidase induced by single-electron photoreduction of intermediate F, generated by reaction of the oxidized enzyme with H(2)O(2), decelerates to an extent compatible with enzyme turnover. (2) In the N139L mutant, the protonic phase of Deltapsi generation coupled to the flash-induced F --> O transition greatly decreases in rate and magnitude and can be assigned to the movement of a proton from E286 to the binuclear site, required for reduction of heme a(3) from the Fe(4+) horizontal lineO(2-) state to the Fe(3+)-OH(-) state. Electrogenic reprotonation of E286 from the inner aqueous phase is missing from the F --> O step in the mutant. (3) In the N139L mutant, the KCN-insensitive rapid electrogenic phase may be composed of two components with lifetimes of approximately 10 and approximately 40 mus and a magnitude ratio of approximately 3:2. The 10 mus phase matches vectorial electron transfer from Cu(A) to heme a, whereas the 40 mus component is assigned to intraprotein proton displacement across approximately 20% of the membrane dielectric thickness. This proton displacement might be triggered by rotation of the charged K362 side chain coupled to heme a reduction. The two components of the rapid electrogenic phase have been resolved subsequently with other D-channel mutants as well as with cyanide-inhibited wild-type oxidase. The finding helps to reconcile the unusually high relative contribution of the microsecond electrogenic phase in the bacterial enzyme ( approximately 30%) with the net electrogenicity of the F --> O transition coupled to transmembrane transfer of two charges per electron.

Inhibition of membrane-bound cytochrome c oxidase by zinc ions: high-affinity Zn2+-binding site at the P-side of the membrane.

In the presence of the uncoupler, external zinc ions inhibit rapidly turnover of cytochrome c oxidase reconstituted in phospholipid vesicles or bound to the membrane of intact mitochondria. The effect is promoted by electron leaks into the oxidase during preincubation with Zn(2+). Inhibition of liposome-bound bovine cytochrome oxidase by external Zn(2+) titrates with a K(i) of 1+/-0.3 microM. Presumably, the Zn(2+)-binding group at the positively charged side is not reactive in the oxidized enzyme, but becomes accessible to the cation in some partially reduced state(s) of the oxidase; reduction of Cu(B) is tentatively proposed to be responsible for the effect.

The fully oxidized form of the cytochrome bd quinol oxidase from E. coli does not participate in the catalytic cycle: direct evidence from rapid kinetics studies.

Cytochrome bd catalyzes the two-electron oxidation of either ubiquinol or menaquinol and the four-electron reduction of O(2) to H(2)O. In the current work, the rates of reduction of the fully oxidized and oxoferryl forms of the enzyme by the 2-electron donor ubiquinol-1 and single electron donor N,N,N',N'-tetramethyl-p-phenylendiamine (TMPD) have been examined by stopped-flow techniques. Reduction of the all-ferric form of the enzyme is 1000-fold slower than required for a step in the catalytic cycle, whereas the observed rates of reduction of the oxoferryl and singly-reduced forms of the cytochrome are consistent with the catalytic turnover. The data support models of the catalytic cycle which do not include the fully oxidized form of the enzyme as an intermediate.

Strong excitonic interactions in the oxygen-reducing site of bd-type oxidase: the Fe-to-Fe distance between hemes d and b595 is 10 A.

Absorption and circular dichroism (CD) spectra of cytochrome bd from Escherichia coli have been compared for the wild type enzyme and an inactive mutant in which a highly conserved E445 in subunit I has been replaced by alanine [Zhang, J., Hellwig, P., Osborne, J. P., Huang, H. W., Moenne-Loccoz, P., Konstantinov, A. A., and Gennis, R. B. (2001) Biochemistry 40, 8548-8556]. The absorption bands of ferrous heme b595 are absent from the spectrum of the dithionite-reduced E445A form of cytochrome bd. The difference between the spectra of the dithionite-reduced WT and E445A enzymes indicates that in the mutant, heme b595 is present but is not reducible by dithionite. Cytochrome bd reveals intense CD signals dominated by heme d, with almost no contribution from heme b595 or heme b558. The CD spectrum of the reduced wild type enzyme in the Soret band indicates strong excitonic interactions between ferrous heme d and ferrous heme b595, and these interactions are not observed in dithionite-reduced E445A mutant, in which heme b595 remains in the ferric state. Modeling the excitonic interactions in both absorption and CD spectra has been carried out, yielding an estimate of the Fe-to-Fe distance between heme d and heme b595 of about 10 A. The physical proximity supports the hypothesis that heme d and heme b595 can form a di-heme oxygen reducing site, a unique structure for respiratory oxidases.

Time-resolved single-turnover of ba3 oxidase from Thermus thermophilus.

The kinetics of the oxidation of fully-reduced ba(3) cytochrome c oxidase from Thermus thermophilus by oxygen were followed by time-resolved optical spectroscopy and electrometry. Four catalytic intermediates were resolved during this reaction. The chemical nature and the spectral properties of three intermediates (compounds A, P and O) reproduce the general features of aa(3)-type oxidases. However the F intermediate in ba(3) oxidase has a spectrum identical to the P state. This indicates that the proton taken up during the P-->F transition does not reside in the binuclear site but is rather transferred to the covalently cross-linked tyrosine near that site. The total charge translocation associated with the F-->O transition in ba(3) oxidase is close to that observed during the F-->O transition in the aa(3) oxidases. However, the P(R)-->F transition is characterized by significantly lower charge translocation, which probably reflects the overall lower measured pumping efficiency during multiple turnovers.

Cytochrome bd from Azotobacter vinelandii: evidence for high-affinity oxygen binding.

Cytochrome bd from Azotobacter vinelandii is a respiratory quinol oxidase that is highly efficient in reducing intracellular oxygen concentration, thus enabling nitrogen fixation under ambient aerobic conditions. Equilibrium measurements of O2 binding to ferrous heme d in the one-electron-reduced form of the A. vinelandii enzyme give Kd(O2) = 0.5 microM, close to the value for the Escherichia coli cytochrome bd (ca. 0.3 microM); thus, both enzymes have similar, high affinity for oxygen. The reaction of the A. vinelandii cytochrome bd in the one-electron-reduced and fully reduced states with O2 is extremely fast approaching the diffusion-controlled limit in water. In the fully reduced state, the rate of O2 binding depends linearly on the oxygen concentration consistently with a simple, single-step process. In contrast, in the one-electron-reduced state the rate of oxygen binding is hyperbolic, implying a more complex binding pattern. Two possible explanations for the saturation kinetics are considered: (A) There is a spectroscopically silent prebinding of oxygen to an unidentified low-affinity saturatable site followed by the oxygen transfer to heme d. (B) Oxygen binding to heme d requires an "activated" state of the enzyme in which an oxygen channel connecting heme d to the bulk is open. This channel is permanently open in the fully reduced enzyme (hence no saturation behavior) but flickers between the open and closed states in the one-electron-reduced enzyme.

Redox control of fast ligand dissociation from Escherichia coli cytochrome bd.

Bacterial bd-type quinol oxidases, such as cytochrome bd from Escherichia coli, contain three hemes, but no copper. In contrast to heme-copper oxidases and similarly to globins, single electron-reduced cytochrome bd forms stable complexes with O(2), NO and CO at ferrous heme d. Kinetics of ligand dissociation from heme d(2+) in the single electron- and fully-reduced cytochrome bd from E. coli has been investigated by rapid mixing spectrophotometry at 20 degrees C. Data show that (i) O(2) dissociates at 78 s(-1), (ii) NO and CO dissociation is fast as compared to heme-copper oxidases and (iii) dissociation in the single electron-reduced state is hindered as compared to the fully-reduced enzyme. Presumably, rapid ligand dissociation requires reduced heme b(595). As NO, an inhibitor of respiratory oxidases, is involved in the immune response against microbial infection, the rapid dissociation of NO from cytochrome bd may have important bearings on the patho-physiology of enterobacteria.

Cytochrome bd, a key oxidase in bacterial survival and tolerance to nitrosative stress.

Cytochrome bd is a quinol respiratory oxidase widely distributed among bacteria, where its expression favours survival under low O2 tensions, and in the presence of nitric oxide (NO) produced by the host immune system. NO reacts with and reversibly inhibits the haem-copper terminal oxidases (HCO) where it binds at the active site, containing a haem-iron and a copper (CuB). NO reacts also similarly with the copper-lacking active site of cytochrome bd, a structural peculiarity that allows one to address the question of whether CuB plays a role in the reaction with NO (and other ligands). In this minireview we discuss the properties of the reactions between bd-type oxidases and NO, and highlight consequences to cell/bacteria physiology.

Single-electron photoreduction of the P(M) intermediate of cytochrome c oxidase.

The P(M)-->F transition of the catalytic cycle of cytochrome c oxidase from bovine heart was investigated using single-electron photoreduction and monitoring the subsequent events using spectroscopic and electometric techniques. The P(M) state of the oxidase was generated by exposing the oxidized enzyme to CO plus O2. Photoreduction results in rapid electron transfer from heme a to oxoferryl heme a3 with a time constant of about 0.3 ms, as indicated by transients at 605 nm and 580 nm. This rate is approximately 5-fold more rapid than the rate of electron transfer from heme a to heme a3 in the F-->O transition, but is significantly slower than formation of the F state from the P(R) intermediate in the reaction of the fully reduced enzyme with O2 to form state F (70-90 micros). The approximately 0.3 ms P(M)-->F transition is coincident with a rapid photonic phase of transmembrane voltage generation, but a significant part of the voltage associated with the P(M)-->F transition is generated much later, with a time constant of 1.3 ms. In addition, the P(M)-->F transition of the R. sphaeroides oxidase was also measured and also was shown to have two phases of electrogenic proton transfer, with tau values of 0.18 and 0.85 ms.