Chemical Modification Variety Oxidation Nitrosylation Dissociation Effects Folding Controls Environmental Reactive

Protein structure - function AA sequence O-N pairing of backbone Ionic/hydrophobic interaction of side chains Chemical environment Ionic strength pH – ie: H+ ions Reactive Side chain modification

Chemistry A + B  AB With total A constant AB increases with either A or B Equilibrium constant Ka=[AB]/([A][B]) With total A constant A/AB switch A/AB indicator Complex chemistry alters sensitivity

Multiple modifications For fixed “A” the amount of product is One “B”: AB Two “B”: AB+AB2 More Hill equation: n: cooperativity Kd: apparent dissociation constant ] [ 1 B K A+AB AB + = Q 2 ] [ ' 1 ) ( B K + - = Q l å + = Q n B K a ] [ 1 n d x Kn + = Q

Chemical sensors 2-3 log dynamic range Decreasing Kd increasing affinity, increases sensitivity Increasing cooperativity increases gain

pH Charged amino acid side chains H+ movement can dramatically alter molecular folding pK=-log( ) [B-][H+] [HB]

Bicarbonate buffers CO2 solubility ~0.03mM/Torr CO2 Hydration Carbonic anhydrase Henderson-Hasselbalch equation K= pK= -log( ) pK = pH – log( ) pH = pK + log( ); pK=6.1 [HCO3-][H+] [CO2] [HCO3-][H+] [CO2] [HCO3-] [CO2] [HCO3-] [CO2]

Bicarbonate buffers [HCO3-][H+] Ka = [CO2] pH 2 pH 7.2 5% CO2 pH 7.4

Open vs Closed Buffer Systems Bicarbonate Physiological pCO2 = 40 mmHg [CO2] = 1.2 mM [CO2]+[HCO3]=31 mM HEPES Equivalent Buffer [HEPES]+[HEPES-]=31 mM [HEPES-]=14.7 [HEPES]=16.3

Open vs Closed Buffers Bicarb Immediate HEPES Immediate Add 10 mM HCl Immediate [HCO3]=20 mM [CO2]=11 mM pH = 6.4 (400 nM) HEPES Add 10 mM HCl Immediate [HEPES-]=4.7 mM [HEPES]=26 mM pH=7.2 (63 nM) Much better pH control near pKa

Open vs Closed Buffer System Bicarb CO2 solubility 1.2 mM [HCO3]=20 mM-350 nM [CO2] = 1.2mM+350 nM pH=7.3 (50 nM) Much better than 6.4 w/o exchange HEPES No mass exchange pH =7.2 Could do bicarb in one step:

pH Control Cell membranes impermeable to H+ Compartmentalization of pH Cytoplasm 7.15 Nucleus 7.2 Mitochondria 8.0 Golgi ~6.3 Lysosome 5.5 Transporters H+/K+ HCO3-/Cl-

Dissociation of amino acid side chains In cytoplasm, pH=7.15, so 80% of histidine is in base form (uncharged). 0.1% of lysine is in its base form. In Golgi, pH=6.3, and 40% of histidine is in base form.

Block NHE Lower intracellular pH stabilize FAs pH control demo Talin-dependent adhesion/motility Glycolysis Block NHE Lower intracellular pH stabilize FAs Talin structure pH dependence of several glycolysis enzymes (Xie et al 2014) Srivastava et al 2008

Reactive modification NO S-nitrosylation Cysteines in hydrophobic acid/base pockets Hemoglobin S-NO forms in oxidative environment Allows NO release in low oxygen Targets vasodilating NO to oxygen starved tissue -S-H -S-N=O

Reactive oxidation Partly reduced oxygen: O2·, H2O2, OH· Protein modification Cys, His, Phe, Tyr, Met Sulfur Ring structures Chain break Cross-linking Chain reaction DNA/Lipid modification

Reactive modification Thymine Thymine glycol Amino acid modification changes local polarity Crosslinking Strand break Thymine glycol distorts DNA structure Kung & Bolton 1997

Electrochemistry Redox reactions describe electron transfer Zn + CuSO4Cu + ZnSO4 Zn + Cu2+Cu + Zn2+ Zn Zn2+ + 2e- and Cu2+ + 2e- Cu 2 GSH + H2O2 GSSG + 2H2O 2 GSH GSSG + 2e- + 2H+ and H2O2 + 2e- + 2H+ 2H2O Inorganic Biological

Electrochemistry-free energy Electrical DG = -nFDE Faraday constant 9.65 104 C/mol Concentration DG = RT ln( Q) Gas constant 8.31 J/K/mol Whole reaction DG = DG0 + RT ln(QProd) – RT ln(Qreac) -nFDE = -nFDE0 + RT ln(Qprod/Qreac) DE = DE0 - RT/nF ln(Qprod/Qreac) Nernst Equation for redox reaction Equilibrium at DG= DE = 0

Electrochemistry-half cells Standard Reduction Potential E0 Metals (Daniell cell) Zn Zn2+ + 2e- Zn2+ + 2e- Zn E0=-0.76V Cu2+ + 2e- Cu E0=+0.34V DE0=0.34-(-0.76) = 1.1V Biological (glutathione) 2 GSH GSSG + 2e- + 2H+ GSSG + 2e- + 2H+  2 GSH E0=+0.18 H2O2 + 2e- + 2H+ 2H2O E0=+1.78 DE0=1.78-(0.18) = 1.6V

Cellular Redox State Biological Steady state trend 2 GSH + H2O2 GSSG + 2H2O DE0= 1.6V DG = -nF (1.6V) + RT ln( ) DE = 1.6V – RT/nF ln( ) Steady state trend 0 = 1.6 –(8.31*310)/(2*9.6e4) ln( ) = 1052 ie: Not a lot of free peroxide in a cell Still needs a catalyst Real cells have many potential half-cells GSSG GSH2 H2O2 GSSG GSH2 H2O2 GSSG GSH2 H2O2 GSSG GSH2 H2O2

GSH:GSSG redox buffer GSH is abundant reducing agent GSSG + 2e- + 2H+  2 GSH E0=+0.18 DE = 0.18 – RT/nF ln( ) DE = 0.18 – 0.03 log( ) GSH:GSSG ratio as marker of redox state More GSH, more negative DE, more reducing GSSG reduction appears as negative in whole reaction Whole reaction more favorable with positive DE More H+, more positive DE, more oxidizing Neutral [H+]2 ~ 10-14 Many biological oxidations include H+ GSSG H2 GSH2 GSSG H2 GSH2

Cellular redox cascade Oxygen radicals are not equivalent ROS generation Mitochondria Photons (UV & ionizing radiation) Inflammatory cells (NADPH oxidase) Radical scavengers O2•-H2O2 superoxide dismutase H2O2H2O Catalase H2O2 + GSH GSSG glutathione peroxidase OH• hydroxyl (uncharged OH-)

Redox state Intracellular reductive Extracellular oxidative Low free oxygen, relatively negative Extracellular oxidative High O2, relatively positive Extracellular antioxidants Extracellular signals that promote oxidative stress Cytoplasmic oxidants Cytoplasmic antioxidants