Steady state kinetics and binding of eukaryotic cytochromes c with yeast cytochrome c peroxidase.
Abstract: 1. The steady state kinetics for the oxidation of ferrocytochrome c by yeast cytochrome c peroxidase are biphasic under most conditions. The same biphasic kinetics were observed for yeast iso-1, yeast iso-2, horse, tuna, and cicada cytochromes c. On changing ionic strength, buffer anions, and pH, the apparent Km values for the initial phase (Km1) varied relatively little while the corresponding apparent maximal velocities varied over a much larger range. 2. The highest apparent Vmax1 for horse cytochrome c is attained at relatively low pH (congruent to 6.0) and low ionic strength (congruent to 0.05), while maximal activity for the yeast protein is at higher pH (congruent to 7.0) and higher ionic strength (congruent to 0.2), with some variations depending on the nature of the buffering ions. 3. Direct binding studies showed that cytochrome c binds to two sites on the peroxidase, under conditions that give biphasic kinetics. Under those ionic conditions that yield monophasic kinetics, binding occurred at only one site. At the optimal buffer concentrations for both yeast and horse cytochromes c, the KD1 and KD2 values approximate the Km1 and Km2 values. At ionic strengths below optimal, binding becomes too strong and above optimal, too weak. 4. Under ionic conditions that are optimal and give monophasic kinetics with horse cytochrome c but are suboptimal for the yeast protein, yeast cytochrome c strongly inhibits the reaction of horse cytochrome c with peroxidase, uncompetitively at one site and competitively at a second site. The appearance of the second site under monophasic conditions is interpreted as an allosteric effect of the inhibitor binding to the first site. 5. The simplest model accounting for these observations postulates two kinetically active sites on each molecule of peroxidase, a high affinity and a low affinity site, that may correspond to the free radical and the heme iron (IV) of the oxidized enzyme, respectively. Both oxidizing equivalents may be discharged at either site. Furthermore, the enzyme appears to exist as an equilibrium mixture of a high ionic strength form, EH and a low ionic strength form, EL, the former reacting optimally with yeast cytochrome c, and the latter with horse cytochrome c.
Publication Date: 1977-02-10 PubMed ID: 14138
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- Journal Article
Summary
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The research article explores the steady state kinetics involved in the oxidation process of ferrocytochrome c (a category of proteins) by yeast cytochrome c peroxidase, analyzing the variations in reaction parameters under different conditions and using different types of cytochromes c.
Study Findings and Interpretations
- The researchers found that under most conditions, the oxidation process displays biphasic kinetics—this means the reaction proceeds in two phases. Different types of cytochromes c were used in the study like yeast iso-1, yeast iso-2, horse, tuna, and cicada, and all demonstrated the same pattern of biphasic kinetics.
- The study reveals important variations with changes in parameters such as ionic strength, buffer anions, and pH levels. Particularly, while the apparent Km values (representing the substrate concentration at half the maximum velocity of reaction) for the initial phase remained relatively consistent, there was a large variation in the corresponding maximal velocities.
- When looking further at the reaction conditions, it was observed that the highest apparent Vmax1 (maximum velocity) for horse cytochrome c is achieved at a relatively low pH (around 6.0) and low ionic strength (around 0.05). In contrast, the yeast protein’s maximal activity occurs at higher pH (around 7.0) and higher ionic strength (around 0.2), but with some changes depending on the type of buffering ions.
- In terms of interaction with peroxidase, the researchers found that cytochrome c seems to bind to two sites on the peroxidase when conditions render biphasic kinetics. However, under conditions yielding monophasic kinetics (single-phase reaction), binding only took place at one site. The binding strength appeared to be too strong below optimum ionic strength, and too weak above.
- An interesting additional find was that under ionic conditions which optimise kinetics for horse cytochrome c (monophasic) but not optimal for the yeast protein, the yeast cytochrome c strongly inhibited the reaction of horse cytochrome c with peroxidase. And this inhibition took place uncompetitively at one site and competitively at another site.
The Proposed Model
- The researchers propose a model that best accounts for their findings. This model suggests the existence of two kinetically active sites on each molecule of peroxidase—one having a high affinity and the other a low affinity. These likely correspond to the free radical and the heme iron (IV) of the oxidized enzyme, respectively.
- The model also posits that both oxidizing equivalents can be discharged at either site. It further suggests that the enzyme exists as an equilibrium mixture of a high ionic strength form (EH) and a low ionic strength form (EL), with EH reacting optimally with yeast cytochrome c, and EL with horse cytochrome c.
Cite This Article
APA
Kang CH, Ferguson-Miller S, Margoliash E.
(1977).
Steady state kinetics and binding of eukaryotic cytochromes c with yeast cytochrome c peroxidase.
J Biol Chem, 252(3), 919-926.
Publication
Researcher Affiliations
MeSH Terms
- Animals
- Binding Sites
- Cytochrome c Group / metabolism
- Cytochrome-c Peroxidase / metabolism
- Grasshoppers
- Horses
- Hydrogen-Ion Concentration
- Isoenzymes / metabolism
- Kinetics
- Osmolar Concentration
- Peroxidases / metabolism
- Protein Binding
- Saccharomyces cerevisiae / enzymology
- Species Specificity
- Tuna
Citations
This article has been cited 15 times.- Payne TM, Yee EF, Dzikovski B, Crane BR. Constraints on the Radical Cation Center of Cytochrome c Peroxidase for Electron Transfer from Cytochrome c. Biochemistry 2016 Aug 30;55(34):4807-22.
- Chreifi G, Hollingsworth SA, Li H, Tripathi S, Arce AP, Magaña-Garcia HI, Poulos TL. Enzymatic Mechanism of Leishmania major Peroxidase and the Critical Role of Specific Ionic Interactions. Biochemistry 2015 Jun 2;54(21):3328-36.
- Page TR, Hoffman BM. Control of cyclic photoinitiated electron transfer between cytochrome c peroxidase (W191F) and cytochrome c by formation of dynamic binary and ternary complexes. Biochemistry 2015 Feb 10;54(5):1188-97.
- Poulos TL. Heme enzyme structure and function. Chem Rev 2014 Apr 9;114(7):3919-62.
- Jasion VS, Doukov T, Pineda SH, Li H, Poulos TL. Crystal structure of the Leishmania major peroxidase-cytochrome c complex. Proc Natl Acad Sci U S A 2012 Nov 6;109(45):18390-4.
- Jasion VS, Poulos TL. Leishmania major peroxidase is a cytochrome c peroxidase. Biochemistry 2012 Mar 27;51(12):2453-60.
- Poulos TL. Thirty years of heme peroxidase structural biology. Arch Biochem Biophys 2010 Aug 1;500(1):3-12.
- Pearl NM, Jacobson T, Arisa M, Vitello LB, Erman JE. Effect of single-site charge-reversal mutations on the catalytic properties of yeast cytochrome c peroxidase: mutations near the high-affinity cytochrome c binding site. Biochemistry 2007 Jul 17;46(28):8263-72.
- Verduyn C, Giuseppin ML, Scheffers WA, van Dijken JP. Hydrogen peroxide metabolism in yeasts. Appl Environ Microbiol 1988 Aug;54(8):2086-90.
- Millett F, Miller MA, Geren L, Durham B. Electron transfer between cytochrome c and cytochrome c peroxidase. J Bioenerg Biomembr 1995 Jun;27(3):341-51.
- Moench SJ, Satterlee JD. A comparison of spectral and physicochemical properties of yeast iso-1 cytochrome c and Cys 102-modified derivatives of the protein. J Protein Chem 1995 Oct;14(7):567-82.
- Osheroff N, Brautigan DL, Margoliash E. Mapping of anion binding sites on cytochrome c by differential chemical modification of lysine residues. Proc Natl Acad Sci U S A 1980 Aug;77(8):4439-43.
- Satterlee JD, Moench S. Proton hyperfine resonance assignments using the nuclear Overhauser effect for ferric forms of horse and tuna cytochrome c. Biophys J 1987 Jul;52(1):101-7.
- Cheung E, Taylor K, Kornblatt JA, English AM, McLendon G, Miller JR. Direct measurements of intramolecular electron transfer rates between cytochrome c and cytochrome c peroxidase: effects of exothermicity and primary sequence on rate. Proc Natl Acad Sci U S A 1986 Mar;83(5):1330-3.
- Dixon DW, Hong X, Woehler SE. Electrostatic and steric control of electron self-exchange in cytochromes c, c551, and b5. Biophys J 1989 Aug;56(2):339-51.
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