High-Pressure Mechanistic Insight Into Bioinorganic NO Chemistry

molecules

Review High-Pressure Mechanistic Insight into Bioinorganic NO Chemistry

Łukasz Orzeł 1, Maria Oszajca 1 , Justyna Polaczek 1 , Dominika Por˛ebska 1 , Rudi van Eldik 1,2,* and Grazyna˙ Stochel 1,*

1 Faculty of Chemistry, Jagiellonian University, Gronostajowa 2, 30-387 Kraków, Poland; [email protected] (Ł.O.); [email protected] (M.O.); [email protected] (J.P.); [email protected] (D.P.) 2 Department of Chemistry and Pharmacy, University of Erlangen-Nuremberg, Egerlandstr 1, 91058 Erlangen, Germany * Correspondence: [email protected] (R.v.E.); [email protected] (G.S.); Tel.: +48-66-777-2932 (R.v.E.); +48-12-686-2502 (G.S.)

Abstract: Pressure is one of the most important parameters controlling the kinetics of chemical reactions. The ability to combine high-pressure techniques with time-resolved spectroscopy has provided a powerful tool in the study of reaction mechanisms. This review is focused on the supporting role of high-pressure kinetic and spectroscopic methods in the exploration of nitric oxide bioinorganic chemistry. Nitric oxide and other reactive nitrogen species (RNS) are important biological mediators involved in both physiological and pathological processes. Understanding molecular mechanisms of their interactions with redox-active metal/non-metal centers in biological targets, such as cofactors, prosthetic groups, and proteins, is crucial for the improved therapy of Citation: Orzeł, Ł.; Oszajca, M.; various diseases. The present review is an attempt to demonstrate how the application of high- Polaczek, J.; Por˛ebska,D.; van Eldik, pressure kinetic and spectroscopic methods can add additional information, thus enabling the R.; Stochel, G. High-Pressure Mechanistic Insight into Bioinorganic mechanistic interpretation of various NO bioinorganic reactions. NO Chemistry. Molecules 2021, 26, 4947. https://doi.org/10.3390/ Keywords: kinetics; high-pressure techniques; nitric oxide; iron complexes; porphyrins; heme molecules26164947 proteins; cobalamins

Academic Editors: Marek Chmielewski, Patryk Niedbala and Maciej Majdecki 1. Introduction With this review, we would like to bring the reader’s attention to high-pressure Received: 30 June 2021 molecular sciences and technology in the advancement of which Professor Janusz Jurczak Accepted: 12 August 2021 made a significant impact, both within synthetic organic chemistry and the development Published: 16 August 2021 of instrumentation [1–3]. Professor Jurczak has been a real leader in this field, and for that reason, the authors of this review dedicate it to his honor. In this review, we focus on Publisher’s Note: MDPI stays neutral the mechanistic studies concerning the interaction of NO with complexes of FeII/III and with regard to jurisdictional claims in CoII/III (including metalloproteins and vitamin B12) performed with the application of published maps and institutional affil- high-pressure kinetic techniques. Since this type of study requires specialized equipment, iations. the number of groups dealing with these studies is limited; therefore, herein, we mainly present the studies performed in our group, as well as those that have resulted from our collaboration with other research groups. Pressure as an experimental variable can provide unique information about the mi- Copyright: © 2021 by the authors. croscopic properties of the studied materials and processes. It can be used as a research Licensee MDPI, Basel, Switzerland. tool for investigating the structure, energetics, dynamics, and kinetics of various processes This article is an open access article and reactions at the molecular level, as well as to chemical synthesis and modifications of distributed under the terms and materials to preserve or improve their quality. The pressure range used for such different conditions of the Creative Commons purposes can extend from a few hundred MPa (reaction kinetics) up to GPa (chemical Attribution (CC BY) license (https:// creativecommons.org/licenses/by/ synthesis or modification of materials). High-pressure kinetic and thermodynamic studies 4.0/). for the elucidation of reaction mechanisms are usually limited to 200 MPa [4–6].

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In order to come as close as possible to the “real” mechanism, it is essential to gain as much information as possible about the chemical understanding of the studied system, empirical rate law, activation and reaction parameters, evidence for possible intermediates, and theoretical analysis of the suggested mechanism. Among the usual variables used in experimental kinetic studies (solvent, ionic strength, pH, etc.), the temperature is a very useful parameter. From the temperature dependence of rate constants and application of the transition state theory, the determination of the enthalpy of activation (∆H‡) and the entropy of activation (∆S‡)—parameters required for the reaction free energy profile— is possible. The additional application of high-pressure kinetic techniques can reveal crucial mechanistic information for chemical processes through the construction of volume profiles with experimentally determined volumes of activation (∆V‡). Such profiles not only complement free energy profiles for the studied reactions but enable a chemical picture to be visualized on the basis of partial molar volume changes that occur along with the reaction coordinate with the estimation of the reaction volume (∆V◦) and the difference in partial molar volumes of product and reactant species [7–9]. From a historical point of view, volumes of activation were obtained for organic reactions long before high-pressure methods were applied to inorganic reactions, for which impressive progress in high-pressure inorganic solution chemistry has been made during the past forty years [10,11]. Furthermore, it was especially the inorganic chemistry community that required experimental techniques to study fast reactions on the second, millisecond, microsecond, and nanosecond timescale. These techniques included stopped- flow, flash-photolysis, pulse-radiolysis, T-jump, and NMR high-pressure developments in the pressure range up to 200 MPa. In this review, we have selected the bioinorganic chemistry of nitric oxide as an example to show how high-pressure kinetic and thermodynamic studies can assist the elucidation of inorganic reaction mechanisms on the molecular level and to improve our understanding of the processes significant for catalysis, health, and the environment. Small inorganic redox molecules, such as nitric oxide (NO), dioxygen (O2), or hydrogen sulfide (H2S), due to their involvement in numerous catalytic, environmental, and biological processes, have been of interest to scientists from various fields of molecular sciences. Among others, all three molecules and their reactive derivatives play diverse roles in various aspects of cell biology and physiology, including metabolism, cellular signaling, and host defense. The bioinorganic chemistry of these redox molecules plays a central role in their biological activity [12–14]. Nitric oxide (NO) is a short-living free radical, and, in contrast to many signaling agents (e.g., various peptides), which rely on receptors where structural relationships determine their function, the chemistry of NO determines its biological roles. There are two distinct reaction types—direct and indirect—which depend on the NO concentration, reactive species formed and reaction kinetics. The direct effects involve interactions of NO itself with biological targets such as redox metal centers, redox-active amino acids, or other radical species. The indirect activity of nitric oxide is mediated by various reactive nitrogen species (RNS). Recognition of the molecular mechanisms for various reaction types by which NO and other RNS can fulfill their biological function is crucial not only for a better understanding of many natural processes but also for planning innovative strategies in health and environmental protection [15–17].

2. High-Pressure Kinetic Techniques Applied to Mechanistic Studies in Coordination Chemistry The fundamental principle behind the concept of performing high-pressure mechanistic studies in coordination chemistry in solution is to gain further mechanistic insight on bond formation and bond cleavage reactions, as well as electron transfer reactions, on the basis of partial molar volume changes. Pressure acceleration is coupled to a negative volume of activation, whereas pressure deceleration is coupled to a positive volume of activation, as illustrated in the following two slides (see Scheme1). Molecules 2021, 26, x FOR PEER REVIEW 3 of 24

Molecules 2021, 26, 4947 3 of 24 volume of activation, whereas pressure deceleration is coupled to a positive volume of activation, as illustrated in the following two slides (see Scheme 1).

A Pressure dependence of reactions in solution B Volume Profile

A -1 A + B Δ Δ ‡ Δ ‡ ln(kp/k0) V = Vf - Vr Δ ‡ mol B Pressure acceleration: - V 3 Δ ‡ Vf ‡ A ----- B Δ ‡ [d(lnk)/dP]T = - V /RT ΔV 0 ‡ Δ ‡ Pressure, MPa ΔV = Activation volume Vr A - B

Δ ‡ Reactants Transition State Products

Pressure deceleration: + V Partial molar volume, cm C Reaction coordinate

A – Cycloaddition; B – Ligand Substitution; C – Homolytic Scission [d(lnk)/dP] = -ΔV ‡/RT T

Scheme 1. (A(A) )Schematic Schematic presentation presentation of ofthe the dependence dependence of ln(k of ln(kp/k0p) /kvs.0 )pressure. vs. pressure. (B) Schematic (B) Schematic presentation presentation of a volume of a volumeprofile. proﬁle.

In the the case case of of reversible reversible reactions, reactions, volume volume profiles profiles can can be be constructed constructed on on the the basis basis of theof the measured measured activation activation volumes volumes for forthe theforwar forwardd and andback back reactions, reactions, or the or reaction the reaction vol- volumeume obtainable obtainable from from the the pressure pressure dependence dependence of of the the overall overall equilibrium equilibrium constant constant K. A few examples of volume profilesprofiles are presented inin thethe subsequentsubsequent sections.sections. During the the 1960s 1960s and and 1970s, 1970s, coordination coordination ch chemistsemists took took the the challenge challenge to develop to develop es- peciallyespecially high-pressure high-pressure instrumentation instrumentation to tost studyudy fast fast reactions reactions on on the the stopped-flow, stopped-flow, T- T- jump, flash-photolysis, flash-photolysis, and NMR timescale. Up until until then, then, high-pressure high-pressure instrumentation was available to study slow reactions as a function of hydrostatic pressure up to 2–3 kbar (200–300 MPa) with a half-life longer than 15 min to pressurize the system and allow for temperature andand pressurepressure equilibration. equilibration. In In principle, principle, one one could could use use a commercial a commercial autoclave auto- withclave awith large a Teflonlarge Teflon bulge filledbulge with filled a with solution, a solution, and withdraw and withdraw samples samples from time from to time to timefollow to thefollow progress the progress of the reaction of the reaction as a function as a function of pressure. of pressure. Different types types of of pressurizing pressurizing systems systems exis exist,t, consisting consisting of a of high-pressure a high-pressure pump, pump, the possibilitythe possibility to change to change from from a hydraulic a hydraulic fluid fluidto another to another pressu pressurizingrizing liquid liquid(such (suchas water as waterfor optical foroptical measurements) measurements) with the with help the of helpa Teflon of a bulge, Teflon pressure bulge, pressure gauges, and gauges, a series and of a pressureseries of pressurevalves, all valves, built into all builta compact into a unit compact for easy unit transportation, for easy transportation, as shown in as Figure shown 1 in Figure1[ 18]. Such transportable units were constructed for use at the Brookhaven [18]. Such transportable units were constructed for use at the Brookhaven National Labor- National Laboratory, New York, for flash-photolysis and pulse-radiolysis experiments, for atory, New York, for flash-photolysis and pulse-radiolysis experiments, for pulse-radiol- pulse-radiolysis experiments at the Hebrew University in Jerusalem, and flash-photolysis ysis experiments at the Hebrew University in Jerusalem, and flash-photolysis experiments experiments at the Jagiellonian University in Krakow, Poland. It enabled the performance at the Jagiellonian University in Krakow, Poland. It enabled the performance of high-pres- of high-pressure kinetic experiments practically all around the world. sure kinetic experiments practically all around the world. An optically see-through two-to-four-window high-pressure cell, as shown in Figure2 , can be used to monitor reaction progress as a function of time, temperature, and pressure for slow (dead time ca. 15 min) and fast (flash-photolysis and pulse-radiolysis) reactions in coordination chemistry [18]. Subsequently, a special technique was developed to fill the pill-box optical cell under un-aerobic conditions [19]. In later developments, the same high-pressure cell, but in a four- window version, was used to perform flash-photolysis and pulse-radiolysis experiments as a function of pressure (see further details given below). In further developments, a very compact high-pressure cell was constructed that enabled spectroscopic studies in the UV–Vis–NIR range up to 400 MPa [20]. In this case the pill-box cell could not be employed any longer, but instead round glass or quartz tubes were used to separate the sample solution from the secondary high-pressure fluid (Figure2C). Depending on the extinction coefficient of the sample to be studied, two different sample tubes with 7.8 mm outer diameter were( used:A) for samples with a high extinction coefficient,(B) a tube with an inner diameter of 1–2 mm was used; for samples with a low extinction coefficient, a tube with an inner diameter of about 5.5 mm was used. In both cases, the tubes were sealed

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volume of activation, whereas pressure deceleration is coupled to a positive volume of activation, as illustrated in the following two slides (see Scheme 1).

A Pressure dependence of reactions in solution B Volume Profile

Δ ‡ Reactants Transition State Products

Pressure deceleration: + V Partial molar volume, cm C Reaction coordinate

A – Cycloaddition; B – Ligand Substitution; C – Homolytic Scission [d(lnk)/dP] = -ΔV ‡/RT T

Scheme 1. (A) Schematic presentation of the dependence of ln(kp/k0) vs. pressure. (B) Schematic presentation of a volume profile.

In the case of reversible reactions, volume profiles can be constructed on the basis of the measured activation volumes for the forward and back reactions, or the reaction volume obtainable from the pressure dependence of the overall equilibrium constant K. A few examples of volume profiles are presented in the subsequent sections. During the 1960s and 1970s, coordination chemists took the challenge to develop especially high-pressure instrumentation to study fast reactions on the stopped-flow, T- jump, flash-photolysis, and NMR timescale. Up until then, high-pressure instrumentation was available to study slow reactions as a function of hydrostatic pressure up to 2–3 kbar (200–300 MPa) with a half-life longer than 15 min to pressurize the system and allow for temperature and pressure equilibration. In principle, one could use a commercial autoclave with a large Teflon bulge filled with a solution, and withdraw samples from time to time to follow the progress of the reaction as a function of pressure. Different types of pressurizing systems exist, consisting of a high-pressure pump, the possibility to change from a hydraulic fluid to another pressurizing liquid (such as water for optical measurements) with the help of a Teflon bulge, pressure gauges, and a series of pressure valves, all built into a compact unit for easy transportation, as shown in Figure 1 Molecules[18].2021, 26Such, 4947 transportable units were constructed for use at the Brookhaven National Labor-4 of 24 atory, New York, for flash-photolysis and pulse-radiolysis experiments, for pulse-radiolysis experiments at the Hebrew University in Jerusalem, and flash-photolysis experiments at the Jagiellonian Universitywith moving in pistons Krakow, to transmit Poland. the It pressure, enabled as the described performance in more detailof high-pres- in the original sure kinetic experimentsmanuscript practically [20]. all around the world.

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Figure 1. (A) Schematic diagram of transportable high-pressure unit complete with high-pressure cell. (a) Pressure medium reservoir, (b) oil reservoir, (c) to vacuum line, (d) one-way valve, (e) holder for pump handles, (f) holder for high-pressure cell, and (g) wheels for easy transportation. Numbers 1–8: (high-pressureA) valves that enable the pressurization of particular(B) sections of the high-pressure unit. (B) Picture of transportable suitcase. Reproduced from Reference [18], with the permission of Figure 1. (A) Schematic diagramAIP Publishing. of transportable high-pressure unit complete with high-pressure cell. (a) Pressure medium reservoir, (b) oil reservoir, (c) to vacuum line, (d) one-way valve, (e) holder for pump handles, (f) holder for high-pressure cell, and (g) wheels for easy transportation.An optically Numbers see-through 1–8: high-pressure two-to-four-window valves that enablehigh-pressure the pressurization cell, as shown of particular in Figure sections of the high-pressure2, unit. can (beB) Pictureused to of monitor transportable reaction suitcase. progress Reproduced as a function from Reference of time, [18 ],temperature, with the permission and pres- of AIP Publishing. sure for slow (dead time ca. 15 min) and fast (flash-photolysis and pulse-radiolysis) reactions in coordination chemistry [18].

(A) (B) (C)

FigureFigure 2. 2. ((AA)) Two-window Two-window opticaloptical high-pressurehigh-pressure cell cell consisting consisting of of 1 cm–thick1 cm–thick quartz quartz windows windows and and sealed sealed by O-by and O- and∆-rings ∆- ringsfor pressures for pressures up to up 200 to MPa. 200 MPa. Usually Usually the pressure the pressure medium medium is water is water and is and thermostated is thermostated by an outerby an copperouter copper coil not coil shown not shownin the diagram.in the diagram. The pill-box The pill-box consists consis of twots of closely two closely fitting fitting quartz quartz cylinders cylinders which which allow allow compression compression of the of solution the solution inside inside the cell. There is a special hole drilled through both cylinders by which the pill-box optical cell can be filled and the cell. There is a special hole drilled through both cylinders by which the pill-box optical cell can be filled and sealed by sealed by turning the two cylinders 180° apart. (B) Four-window optical high-pressure cell with a thermostating copper turning the two cylinders 180◦ apart. (B) Four-window optical high-pressure cell with a thermostating copper coil. (C) Two coil. (C) Two separated cylinders of the pill-box optical cell that make a gastight fit. Reproduced from Reference [18], with theseparated permission cylinders of AIP of Publishing. the pill-box optical cell that make a gastight fit. Reproduced from Reference [18], with the permission of AIP Publishing. Subsequently, a special technique was developed to fill the pill-box optical cell under The limitations in terms of a faster timescale could only be overcome by turning to un-aerobic conditions [19]. In later developments, the same high-pressure cell, but in a stopped-flow techniques by which reactants could be mixed rapidly. This required the four-window version, was used to perform flash-photolysis and pulse-radiolysis experi- construction of a complete stopped-flow device within a high-pressure cell. The details ments as a function of pressure (see further details given below). In further developments, of this construction are shown in Figure3[ 21]. This high-pressure stopped-flow unit was a very compact high-pressure cell was constructed that enabled spectroscopic studies in designed in such a way that the sample syringes contained ca. 2 mL of solution but enabled the UV–Vis–NIR range up to 400 MPa [20]. In this case the pill-box cell could not be em- more than 20 kinetic traces to be recorded with a single filling of the sample syringes [21]. ployed any longer, but instead round glass or quartz tubes were used to separate the sample solution from the secondary high-pressure fluid (Figure 2C). Depending on the extinction coefficient of the sample to be studied, two different sample tubes with 7.8 mm outer diameter were used: for samples with a high extinction coefficient, a tube with an inner diameter of 1–2 mm was used; for samples with a low extinction coefficient, a tube with an inner diameter of about 5.5 mm was used. In both cases, the tubes were sealed with moving pistons to transmit the pressure, as described in more detail in the original manuscript [20]. The limitations in terms of a faster timescale could only be overcome by turning to stopped-flow techniques by which reactants could be mixed rapidly. This required the construction of a complete stopped-flow device within a high-pressure cell. The details of this construction are shown in Figure 3 [21]. This high-pressure stopped-flow unit was designed in such a way that the sample syringes contained ca. 2 mL of solution but enabled more than 20 kinetic traces to be recorded with a single filling of the sample syringes [21]. At the start of the measurements, the receiver syringe was filled with solvent and

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fully emptied by flushing the optical path in an upward direction. The filled sample syringes were then mounted;At the start atof the the end measurements, of the experiment, the receiver the syringe sample was syringes filled with were solvent empty, and fully and the receiver syringeemptied was by flushingfilled with the opticalthe product path in mixture. an upward Under direction. favorable The filled conditions, sample syringes it was possible to werecomplete then mounted; a whole at pressure the end of dependence the experiment, consisting the sample of syringes four werepressures, empty, and between 5 and 150 theMPa, receiver and syringefive kinetic was filled expe withriments the product at each mixture. pressure. Under The favorable dead-time conditions, of it was possible to complete a whole pressure dependence consisting of four pressures, the stopped-flow unit was determined to be 100 ms from the time the step-motor was between 5 and 150 MPa, and five kinetic experiments at each pressure. The dead-time triggered to the timeof thecomplete stopped-flow mixing unit of was the determined reactants towas be 100achieved. ms from the time the step-motor was triggered to the time complete mixing of the reactants was achieved.

(A) (B) (C)

FigureFigure 3. ( A3.) Cross-sectional(A) Cross-sectional diagram diagram of the high-pressure of the high-pressu vessel andre stopped-flow vessel and apparatus. stopped-flow (1) Pressure apparatus. vessel lid,(1) (2)Pressure pressure vessel, vessel (3) lid, high-pressure (2) pressure window vessel, seal (3) assembly, high-pressure (4) high-pressure window sapphire seal window,assembly, (5) step(4) motor,high-pressure (6) screw- drivesapphire mechanism, window, (7) drive (5) shaft, step (8)motor, syringe (6) thrust screw-drive plate, (9)reactant mechanism, syringe (7) pistons, drive (10) shaft, reactant (8) syringe syringes, thrust (11) Kel-F plate, cell block,(9) reactant (12) mixing syringe jet, (13) receiverpistons, syringe, (10) re andactant (14) receiver-syringesyringes, (11) piston.Kel-F (cellB) High-pressure block, (12) mixing stopped-flow jet, (13) instrument receiver as constructedsyringe, byand van (14) Eldik receiver-syringe and co-workers [21 piston.]. Essential (B) High-pressure components, such stopped-flow as syringes and instrument mixing chamber. as constructed (C) Complete unitby attached van Eldik to the and lid ofco-workers the pressure [21]. cell. ReproducedEssential components, from Reference such [21], withas syringes the permission and mixing of AIP Publishing. chamber. (C) Complete unit attached toThe the next lid developmentof the pressure was cell. the applicationReproduced of pulsed-laserfrom Reference flash-photolysis. [21], with the In this permission of AIP Publishing.case, a four-window high-pressure cell was used in a perpendicular way to flash and trigger the optical detection system after a short time delay in the nanosecond time range [18]. The next developmentFlash photolysis was isthe a powerful application technique of pulsed-laser to study the reaction flash-photolysis. mechanism of coordinationIn this case, a four-windowand high-pressure organometallic cell compounds was used in solution,in a perpendicular where the pressure way to variable flash and adds trig- a further dimension to the mechanistic analysis in terms of volume changes associated with the ger the optical detectionformation system of the after transition a short state. time A laser-pulse delay in the disturbs nanosecond the existing time equilibrium range [18]. situation, Flash photolysis is ona powerful which the subsequenttechnique recombination to study the reaction reaction allows mechanism to follow the of kinetics coordination of the studied and organometallicreaction compounds on a micro- in tosolution, nanosecond wh timescale.ere the pressure A typical laser-pulsed variable adds flash photolysisa further setup dimension to the mechanisticis shown in Figure analysis4. in terms of volume changes associated with the A further development was the challenge to perform pulse-radiolysis experiments at formation of the transitionelevated pressurestate. A to laser-puls study the formatione disturbs of the radicals existing that can equilibrium undergo ligand situation, substitution on which the subsequentand electron recombination transfer reactions. reaction This wasallows not an to easy follow task, the since kinetics the optical of quartzthe stud- windows ied reaction on a micro-used in to the nanosecond standard high-pressure timescale. cell A did typical not allow laser-pulsed an electron beam flash to photolysis reach the sample setup is shown in Figurewithin the4. pill-box cell. Therefore, we had to construct a special steel window that consisted

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of a single 8 mm hole that was 13.3 mm deep, in combination with seven 2.3 mm holes at Molecules 2021, 26, x FOR PEER REVIEW4.3 mm deep, to form a metal grid of 0.7 mm steel that enables the electron beam to enter 6 of 24 the high-pressure cell [22]. The construction of the steel window was such that it met the pill-box cell on the ﬂat optical window, allowing for very close contact with the pressure medium. The details of this construction are shown in Figure5A,B.

Figure 4. A four-window high-pressure cell built into a pulsed-laser flash photolysis unit with an optical detection system, using the pill-box cell shown in Figure 2, which allows a perpendicular alignment of the laser beam and optical detection system.

A further development was the challenge to perform pulse-radiolysis experiments at elevated pressure to study the formation of radicals that can undergo ligand substitution and electron transfer reactions. This was not an easy task, since the optical quartz windows used in the standard high-pressure cell did not allow an electron beam to reach the sample within the pill-box cell. Therefore, we had to construct a special steel window that consisted of a single 8 mm hole that was 13.3 mm deep, in combination with seven 2.3 mm holes at 4.3 mm deep, to form a metal grid of 0.7 mm steel that enables the electron beam to enter the high-pressure cell [22]. The construction of the steel window was such that it metFigure Figurethe pill-box 4. A four-window4. cellA four-windowon the high-pressure flat optical cell builtwindhigh-pressure intoow, a pulsed-laserallowing for ﬂashcell very photolysis built close unitinto contact with a an withpulsed-laser the flash photolysis unit with an pressureoptical medium. detection system, The details using the of pill-boxthis construction cell shown in Figureare shown2, which in allows Figure a perpendicular 5A,B. alignmentoptical of thedetection laser beam and system, optical detection using system. the pill-box cell shown in Figure 2, which allows a perpendicular alignment of the laser beam and optical detection system.

(Asample) within the pill-box cell. Therefore,(B) we had to construct a special steel window that

Figure 5.Figure (A) Schematic 5. (A) Schematic diagram diagram consistedof the of theelectron electron beam of beam awindow window single for high-pressurehi 8gh-pressure mm hole pulse pulse radiolysis. that radiolysis. was The electronThe 13 electron.3 beam mm enters beam deep, enters in combination with seven 2.3 mm from thefrom right the side. right side.(B) Schematic (B) Schematicholes diagram diagram at of of 4.3the the pill-boxpill-box mm sample sampledeep, cell, cell, showing to showing form the electron the a electronmetal beam and beam lightgrid and beam oflight paths 0.7 beam and mm paths steel that enables the electron beam and thethe irradiated irradiated volume. volume. Reproduced from from Reference Reference [22], [22], with with the permission the permission of AIP Publishing.of AIP Publishing. to enter the high-pressure cell [22]. The construction of the steel window was such that it The system described here was used for all pulse radiolysis experiments performed at The system described here was used for all pulse radiolysis experiments performed Brookhavenmet the National pill-box Laboratory, cell New on York, the using flat the optical Laser Electron wind Acceleratorow, Facility allowing for very close contact with the at Brookhaven(LEAF), and National the Hebrew Laboratory, University New in Jerusalem York, using [22]. Typicalthe Laser kinetic Electron traces Accelerator showing Fa- cilitythatpressure (LEAF), the electron and themedium. beam Hebrew indeed University reaches The the details formamidein Jerusalem of sample this [22]. within Typicalconstruction the kinetic pill-box traces cell areare showing shown in Figure 5A,B. thatpresented the electron in Figure beam6 on indeed a nanosecond reaches timescale. the formamide sample within the pill-box cell are presented in Figure 6 on a nanosecond timescale.

(A) (B) Figure 5. (A) Schematic diagram of the electron beam window for high-pressure pulse radiolysis. The electron beam enters from the right side. (B) Schematic diagram of the pill-box sample cell, showing the electron beam and light beam paths and the irradiated volume. Reproduced from Reference [22], with the permission of AIP Publishing.

The system described here was used for all pulse radiolysis experiments performed at Brookhaven National Laboratory, New York, using the Laser Electron Accelerator Fa- cility (LEAF), and the Hebrew University in Jerusalem [22]. Typical kinetic traces showing that the electron beam indeed reaches the formamide sample within the pill-box cell are presented in Figure 6 on a nanosecond timescale.

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((AA)) ((BB)) FigureFigureFigure 6.6. 6. (( AA(A)) ) TypicalTypical Typical kinetickinetic kinetic tracestraces traces toto to showshow show thethe the accelerationacceleration acceleration ofof of theth thee reactionreaction reaction atat at 100100 100 MPaMPa MPa inducedinduced induced byby by thethe the LEAFLEAF LEAF facilityfacility facility atat at BNL,BNL, BNL, ‡ NewNewNew York.York. York. (( BB(B)) )PlotPlot Plot ofof of lnln lnkkk versus versus pressure, pressure, where where Δ∆ ΔVV‡‡was was calculated calculated from from from the the the slope slope slope of of of the the the linear linear linear dependence dependence dependence on on on pressure. pressure. pressure.

OurOurOur next nextnext challenge challengechallenge was waswas to to constructto constructconstruct a high-pressure aa hihigh-pressuregh-pressure probe probeprobe for a for Brukerfor aa BrukerBruker 400 MHz 400400 NMR MHzMHz machineNMRNMR machine machine with a with widewith a a bore wide wide magnet bore bore magnet magnet and a 9and and cm a cylindricala 9 9 cm cm cylindrical cylindrical bore. The bore. bore. wide-bore The The wide-bore wide-bore probe couldprobe probe 1 131 1513 1715 17 31 31 becouldcould tuned be be tuned fortuned various for for various various isotopes, isotopes, isotopes, such as such suchH, as as C, H,1H, 13N,C,C, 15N,O,N, and17O,O, and andP, with31P,P, with with a pressure a a pressure pressure range range range of 200ofof 200 200 MPa. MPa. MPa. The The The construction construction construction of of thisof this this probe probe probe was was was completed completed completed in in in 1994 1994 1994 (see (see (see Figure Figure Figure7 )[7) 7) 23[23]. [23].]. TenTen Ten yearsyearsyears later,later, later, wewe we werewere were successful successful successful to to to build build build a a narrow-borea narrow-bore narrow-bore high-pressure high-pressure high-pressure probe probe probe that that that could could could ﬁt fit afit standardaa standard standard magnet magnet magnet with with with a a 3.5a 3.5 3.5 cm cm cm cylindrical cylindrical cylindrical bore bore bore (see (see (see Figure Figure Figure8)[ 8) 8) 24[24]. [24].]. TheThe The uniqueunique unique possibilitypossibility possibility tototo exchangeexchange exchange normalnormal normal andand and high-pressurehigh-pressure high-pressure probesprobes probes fromfrom from thethe the bottombottom bottom ofof of thethe the NMRNMR NMR magnet,magnet, magnet, mademade made ititit easyeasy easy toto to performperform perform high-pressure high-pressure high-pressure measurements measurements measurements since since since everything everything everything could could could be be tunedbe tuned tuned in termsin in terms terms of temperature,ofof temperature, temperature, frequency frequency frequency and and and pressure pressure pressure from from from the bottomthe the bottom bottom of the of of magnet.the the magnet. magnet. The BrukerThe The Bruker Bruker Company Com- Com- alsopanypany now also also has now now a high-pressurehas has a a high-pressure high-pressure unit available unit unit av availableailable for their for for range their their ofrangerange NMR of of machines.NMR NMR machines. machines.

FigureFigureFigure 7.7. 7. SchematicSchematic Schematic diagramdiagram diagram ofof of thethe the titaniumtitanium titanium high-pressurehigh-pre high-pressuressure NMRNMR NMR probe-headprobe-head probe-head designeddesigned designed for for aa a wide-widewide- boreborebore magnet.magnet. magnet. ReproducedReproduced Reproduced fromfrom from ReferenceReference Reference [[23],23 [23],], withwith with thethe the permissionpermission permission ofof of AIPAIP AIP Publishing.Publishing. Publishing.

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Molecules 2021, 26, x FOR PEER REVIEW 8 of 24

(A) (B) Figure 8. (A) Cross-sectional view of the high-pressure autoclave with top and bottom plug, sample Figure 8. (A) Cross-sectional view of the high-pressure autoclave with top and bottom plug, sample tube, macor plug and sample coil. (B) Photograph of two narrow bore probe heads: (a) aluminum jackettube, sealing macor the plug double and helix sample used coil. for thermostating, (B) Photograph (b) ofhigh-pressure two narrow vessel, bore (c) probe platform heads: carrying (a) aluminum thejacket autoclave, sealing (d) the capacitors, double helix (e) capacitor used for platfo thermostating,rms, (f) tuning (b) high-pressure rods, (g) high-pressure vessel, (c) connector, platform carrying (h)the thermocouple, autoclave, (d) (i) capacitors, BNC connector, (e) capacitor (j) Pt-100 platforms, connector, (f)(k) tuning copper rods,tubing, (g) and high-pressure (l) wide-bore connector,

(h)adapter. thermocouple, Reproduced (i) from BNC Reference connector, [24], (j) with Pt-100 the connector, permission (k) of copperAIP Publishing. tubing, and (l) wide-bore adapter. (A) (B) Reproduced from Reference [24], with the permission of AIP Publishing. Figure 8.Finally, (A) Cross-sectional a PhD view student of the high-pressure at the University autoclave with of top Frankfurt and bottom plug,am sampleMain constructed a joule- tube,heating macor plugtemperature-jump and sample coil. (B) Photograph cell that of enabled two narrow a bore discharge probe heads: of (a) 20 aluminum kV to “jump” the tempera- Finally, a PhD student at the University of Frankfurt am Main constructed a joule- jacketture sealing of the the solutiondouble helix by used 2–3 for thermostating, °C, followed (b) high-pressure by the relaxation vessel, (c) platform of the carrying studied equilibrium to the theheating autoclave, temperature-jump(d) capacitors, (e) capacitor platfo cellrms, that (f) enabledtuning rods, a(g) discharge high-pressure ofconnector, 20 kV to “jump” the temperature (h)new thermocouple, temperature (i) BNC for connector, pressures◦ (j) Pt-100 up connector, to 200 MPa(k) copper [25]. tubing, The and high-pressure (l) wide-bore cell itself is shown adapter.inof Figure the Reproduced solution 9A, whereas from by Reference 2–3 the [24],C, temperature-jump followedwith the permission by the of AIPcell relaxation Publishing. is shown ofin Figure the studied 9B. The equilibrium latter cell was to the new temperature for pressures up to 200 MPa [25]. The high-pressure cell itself is shown in constructedFinally, a PhD from student Kel-F at the and University had two of opticalFrankfurt wi amndows Main constructed for the photometric a joule- detection of the heatingabsorbanceFigure temperature-jump9A, changes whereas cellas the thata function temperature-jumpenabled a of discharge time, and of 20 twokV cell to Teflon “jump” is shown membranesthe tempera- in Figure by9 whichB. The the latter pres- cell was turesureconstructed of thewas solution transmitted by from 2–3 °C, Kel-F to followed the andinside by th had eof relaxation the two Kel-F opticalof the cell. studied Gold windows equilibrium electrodes for to the were the photometric used for the rapid detection of new temperature for pressures up to 200 MPa [25]. The high-pressure cell itself is shown indischargethe Figure absorbance 9A, whereas of 20 thekV. changes temperature-jump The high-pressure as a function cell is shown cell of inwa time,Figures mounted 9B. and The twolatter on cell Teﬂona standard was membranes base plate by of whicha the constructedpressureMessanlagen from was Kel-F Studiengesellschaft transmitted and had two optical to the wi (Göttingen,ndows inside for ofthe thephotometricGermany) Kel-F detection cell. T-jump Gold of theinstrument. electrodes In were conclu- used for the absorbancesion,rapid it dischargecan changes be said as a of functionthat 20 very kV. of time, Thesimilar and high-pressure twoeffect Teflons are membranes observed cell was by withwhich mounted a the joule-heating pres- on a standard temperature- base plate of a surejump was transmittedsystem than to the with inside aof laser-heatedthe Kel-F cell. Gold analog electrodes when were inorganicused for the rapidcomplex formation pro- dischargeMessanlagen of 20 kV. The Studiengesellschaft high-pressure cell was (Göttingen,mounted on a standard Germany) base plate T-jump of a instrument. In conclusion, Messanlagencessesit can beof theStudiengesellschaft said type that investigated very (Göttingen, similar are effects studied.Germany) are T-jump observed instrument. with In aconclu- joule-heating temperature-jump sion, it can be said that very similar effects are observed with a joule-heating temperature- jumpsystem system than than with with a laser-heated a laser-heated analog when analog inorganic when complex inorganic formation complex pro- formation processes of cessesthe of type the type investigated investigated are are studied. studied.

(A) (B) Figure 9. (A) High-pressure (temperature-jumpA) cell as designed by Doss et al. High-pressure(B) cell: Figure 9. (A) High-pressure temperature-jump cell as designed by Doss et al. High-pressure cell: A—vesselFigure lid,9. (B—vesselA) High-pressure body, C—steel temperature-jump piston, D—window support, cell as E— designed∆- and O-rings, by G—spaceDoss et al. High-pressure cell: A—vessel lid, lid, B—vessel B—vessel body, body, C—steel C—steel piston, piston, D—window D—window support, support, E—∆ E—- and∆ -O-rings, and O-rings, G—space G—space for T-jump cell, H—∆- and O-ring, J—steel high-voltage connector, K—insulation material, L—circulation coil, and M—high-voltage plug. (B) T-jump cell: 1—upper electrode, 2—deaeration hole, 3—electrode thread support, 4—Kel-F cell body, 5—membrane support, 6—Teﬂon membrane, 7—optical window, 8—lower electrode, 9—threaded bolt, and 10—support ring. Reproduced from Reference [25], with the permission of AIP Publishing. Molecules 2021, 26, 4947 9 of 24

3. NO Binding to Heme Proteins and Model Iron Porphyrin Complexes Mechanistic studies on the NO binding to iron(II) and iron(III) porphyrin model systems, as well as heme proteins, provided answers to numerous questions related to the influence of the radical character of NO on its reaction mechanism with an iron center, the role of the metal–ligand system, the role of the amino acid residues surrounding the active center, and the protein superstructure. The activation volume parameter (∆V‡), determined in high-pressure stopped-flow or laser-flash photolysis studies, provided information that was particularly important in the process of unraveling the reaction mechanisms of formation and decay of nitrosyl hemoproteins. Ford and co-workers [26] performed systematic hydrostatic pressure (0.1–250 MPa) measurements and determined ∆V‡ parameters for the “on” and “off” reactions with ferric, as well as for “on” reactions with ferrous water-soluble porphyrins (Table1), where TPPS represents the tetrakis(4-sulfonatophenyl)porphinato anion, and TMPS represents the tetrakis(sulfonatomesityl)porphinato anion (Table1).

Table 1. Rate constants and activation volumes for the reactions of selected iron porphyrins and heme proteins with NO.

∆V‡ k (M−1 s−1) ∆V‡ (cm3/mol) k (s−1) off Reference on on off (cm3/mol) III 4− 5 (at 25 ◦C) 2 (at 25 ◦C) Fe (TPPS )(H2O)2 4.5 × 10 +9 ± 1 5 × 10 +18 ± 2 [26] III 4− 6 (at 25 ◦C) 2 (at 25 ◦C) Fe (TMPS )(H2O)2 2.8 × 10 +13 ± 1 9 × 10 +17 ± 3 [26] ◦ ◦ FeIII(TMPS4−)(OH) 7.4 × 103 (at 10 C) −16.2 ± 0.4 1.5 (at 10 C) +7.4 ± 1.0 [27] III 8− 5 (at 25 ◦C) 2 (at 25 ◦C) Fe (P )(H2O)2 8.2 × 10 +6.1 ± 0.1 2 × 10 +16.8 ± 0.4 [28] ◦ ◦ FeIII(P8−)(OH) 5.1 × 104 (at 25 C) −6.1 ± 0.2 11 (at 25 C) +17 ± 3 [28] III 8+ 4 (at 25 ◦C) (at 25 ◦C) Fe (P )(H2O)2 1.5 × 10 +1.5 ± 0.3 26 +9.3 ± 0.5 [29] III 8+ 3 (at 25 ◦C) (at 25 ◦C) Fe (P )(H2O)(OH) 1.6 × 10 −13.8 ± 0.4 6.2 +2.6 ± 0.2 [29] II 4− 9 (at 25 ◦C) Fe (TPPS )(H2O)2 1.5 × 10 +5 ± 1 - - [26] II 4− 9 (at 25 ◦C) Fe (TMPS )(H2O)2 1.0 × 10 +2 ± 1 - - [26] ◦ ◦ metMb 4.8 × 104 (at 25 C) +21 ± 1 29 (at 25 C) +16 ± 1 [30] 5 (at 25 ◦C) (at 25 ◦C) P450cam—resting state 3.2 × 10 +28 ± 2 0.35 +31 ± 1 [31] 6 (at 25 ◦C) (at 25 ◦C) P450cam—camphor 3.2 × 10 −7.3 ± 0.2 1.93 +24 ± 1 [31] ◦ ◦ SR complex in methanol 2.7 × 106 (at 25 C) +6 ± 1 1.8 (at 25 C) -[32] − R-SO complex in ◦ ◦ 3 6 × 104 (at 25 C) −21 ± 4 2.2 × 103 (at 25 C) +7 ± 3 [33] methanol − 6 (at 25 ◦C) 4 (at 25 ◦C) R-SO3 complex in toluene 1.8 × 10 −25 ± 4 1.2 × 10 +7 ± 3 [33]

Molecules 2021, 26, x FOR PEER REVIEW Volume proﬁles that clearly visualize the position of the transition state can be10 con- of 24 structed based on the obtained activation volumes. Examples of volume proﬁles constructed for the ferric TMPS porphyrin are presented in Figure 10 as a function of pH.

(A) (B)

III III IIIIII FigureFigure 10. 10.Volume Volume proﬁles profiles for for reversible reversible NO NO binding binding to to Fe(TMPS) Fe(TMPS) (A ()A (TMPS)Fe) (TMPS)Fe(H(H2O)2O)2 and2 and (B ()B (TMPS)Fe) (TMPS)Fe (OH)(OH) under under acidicacidic and and alkaline alkaline conditions, conditions, respectively. respectively.

For both ferri– and ferro–diaqua–heme models, positive ΔV‡on values were determined (Table 1) [26]. Due to the six-coordinate nature of ferri–heme complexes, the large positive ΔV‡on values provided support for a dissociatively activated mechanism with the limiting step expressed by Equation (1).

III ⎯⎯ III (Por)Fe (H2O)2 ⎯⎯ (Por)Fe (H2O) + H2O (1)

III ⎯⎯ II + (Por)Fe (H2O) + NO ⎯⎯ (Por)Fe (H2O)(NO ) + H2O (2) Further support for this conclusion was provided by high-pressure NMR studies on the water-exchange kinetics of the diaqua ferric porphyrins, performed in the van Eldik group [34], which confirmed that the activation parameters for the nitrosylation reaction of (Por)FeIII are largely defined by a dissociative mechanism. The mentioned studies allowed concluding that the lability of coordinated water plays a crucial role in the dynamics of the reversible NO binding to ferrous porphyrins, whereas the free radical nature of NO has only a minor (if any) influence at all. This conclusion was further supported by detailed studies with the application of highly negatively charged, (Por8−)FeIII, and positively charged, (Por8+)FeIII, water-soluble porphyrins (Figure 11) [28,29]. The determined activation parameters, pointed towards dissociative or interchange dissociative mechanism for NO binding to (Por8−)FeIII and interchange dissociative (Id) or even interchange (I) for (Por8+)FeIII (Table 1). This was reflected by much higher ΔV‡on values obtained for the nitrosylation of (Por8−)FeIII than for (Por8+)FeIII, for which substantially smaller ΔV‡on values were found. These studies revealed that the nature and charge of the substituents on the porphyrin ring tune the lability of coordinated water by affecting the electron density present on the iron(III) center. In porphyrins with electron-donating substituents, breakage of the Fe-H2O bond is facilitated, whereas an opposite effect (bond stabilization) is a consequence of electron-withdrawing substituents. Such properties result in the observed change in the mechanism of NO binding from predominantly dissociative for the (Por8−)FeIII to interchange for (Por8+)FeIII [28,29].

Molecules 2021, 26, 4947 10 of 24

‡ For both ferri– and ferro–diaqua–heme models, positive ∆V on values were determined (Table1)[ 26]. Due to the six-coordinate nature of ferri–heme complexes, the large ‡ positive ∆V on values provided support for a dissociatively activated mechanism with the limiting step expressed by Equation (1).

k III →1 III (Por)Fe (H2O)2 ← (Por)Fe (H2O) + H2O (1) k−1

k III →2 II + (Por)Fe (H2O) + NO ← (Por)Fe (H2O)(NO ) + H2O (2) k−2 Further support for this conclusion was provided by high-pressure NMR studies on the water-exchange kinetics of the diaqua ferric porphyrins, performed in the van Eldik group [34], which confirmed that the activation parameters for the nitrosylation reaction of (Por)FeIII are largely defined by a dissociative mechanism. The mentioned studies allowed concluding that the lability of coordinated water plays a crucial role in the dynamics of the reversible NO binding to ferrous porphyrins, whereas the free radical nature of NO has only a minor (if any) influence at all. This conclusion was further supported by detailed studies with the application of highly negatively charged, (Por8−)FeIII, and positively charged, (Por8+)FeIII, water-soluble porphyrins (Figure 11)[28,29]. The determined activation parameters, pointed towards dissociative or interchange dissociative mechanism for NO binding to (Por8−)FeIII and 8+ III interchange dissociative (Id) or even interchange (I) for (Por )Fe (Table1). This was ‡ 8− III reflected by much higher ∆V on values obtained for the nitrosylation of (Por )Fe than 8+ III ‡ for (Por )Fe , for which substantially smaller ∆V on values were found. These studies revealed that the nature and charge of the substituents on the porphyrin ring tune the lability of coordinated water by affecting the electron density present on the iron(III) center. In porphyrins with electron-donating substituents, breakage of the Fe-H2O bond is facilitated, whereas an opposite effect (bond stabilization) is a consequence of electron- withdrawing substituents. Such properties result in the observed change in the mechanism of NO binding from predominantly dissociative for the (Por8−)FeIII to interchange for (Por8+)FeIII [28,29]. The revealed mechanism made it easier to explain the NO reactivity towards iron(II) porphyrins, for which the rate constants were determined to be about three orders of magnitude larger than for its ferric analogues (Table1). It became clear that due to the five-coordinate nature of high-spin iron(II) porphyrins, no limitations related to ligand ‡ substitution occurred. Small, positive ∆V on values determined for NO binding to iron(II) porphyrins allowed to classify the mechanism as diffusion-limited. In such a mechanism, an encounter complex [(Por)FeII||(NO)] creation before metal–ligand bond formation is ‡ ‡ the rate-limiting step (ka k−D), thus ∆V on = ∆V D (activation volume for diffusion) (3) [26]. k II →D II ka II (Por)Fe + NO ← [(Por)Fe ||(NO)] → (Por)Fe (NO) (3) k−D This is in agreement with the fact that positive activation volumes for diffusion in various solvents are a result of the viscosity increase with increasing pressure [35]. The dynamics of NO binding to FeII porphyrins are different than for CO binding. Several orders of magnitude lower second-order rate constants for CO coordination, together with ‡ the negative ∆V on, provide confirmation that in this case, an activation-limited mechanism ‡ ‡ ‡ exists (k−D ka). In the activation-limited mechanism (∆V on = ∆V a), the negative ∆V on value is determined by FeII-L bond formation and concomitant change in spin state. It was surprising to observe similar NO binding dynamics for pentacoordinate ferric- monohydroxo TMPS [27]. In this case, much lower reaction rates, concomitant with ‡ negative ∆V on allowed to conclude that the reactivity pattern is not always controlled by the lability of the iron(III) center (as would be expected in the case of the diffusion- limited reactions), but can be determined by the spin density reorganization and accom- Molecules 2021, 26, 4947 11 of 24

Molecules 2021, 26, x FOR PEER REVIEW 11 of 24 panying structural changes [27]. Comparison of typical volume proﬁles determined for six-coordinate diaqua vs. ﬁve-coordinate monohydroxo iron(III) porphyrins are presented in Figure 10.

R – para-t-butyl-C6H4

(A) (B)

FigureFigure 11. 11. StructureStructure of of (A (A) )(Por (Por8+8+)Fe)FeIIIIII andand (B (B) )(Por (Por8−8)Fe−)FeIII.III Reprinted. Reprinted with with permission permission from InorganicInorganic Chemistry Chemistry, 2005, 2005,, 44 ,44, 7717–77317717–7731 Copyright Copyright 2005 2005 AmericanAmerican Chemical Chemical Society Society and and Inorganic Inorganic Chemistry Chemistry, 2006, 2006,, 45, 1326–1337. 45, 1326–1337. Copyright Copyright 2006 American2006 Amer- icanChemical Chemical Society Society [28 [28,29].,29].

TheComparison revealed mechanism of the results made obtained it easier for the to ex modelplain porphyrins the NO reactivity with the towards ones for iron(II) ferri- porphyrins,heme proteins for which was crucial the rate to reveal constants the role were of determined an amino acid tochain be about surrounding three orders theactive of mag- nitudecenter: larger polarity than of for the its protein ferric pocket,analogues hydrogen-bonding (Table 1). It became interactions, clear that prosthetic due to groupthe five- coordinatesolvent-accessibility, nature of high-spin etc. The first iron(II) high-pressure porphyrins, studies no limitations concerning reversiblerelated to NOligand binding substi- by heme protein were performed for met-myoglobin (metMb). A successful collaboration of tution occurred. Small, positive ΔV‡on values determined for NO binding to iron(II) por- three research groups (G. Stochel, R. van Eldik and P. Ford) resulted in a joint publication re- phyrins allowed to classify the mechanism as diffusion-limited. In such a mechanism, an porting high-pressure effects on the nitrosylation reaction rates [30]. The semi-independent encounter complex [(Por)FeII||(NO)] creation before metal–ligand bond formation is the results were performed by using two techniques: laser flash-photolysis and stopped-flow. rate-limiting step (𝑘 ≫𝑘 ), thus ΔV‡on = ΔV‡D (activation volume for diffusion) (3) [26]. The application of these two fundamentally different techniques resulted in the determination of a set of activation parameters being in good agreement and providing insight into II ⎯⎯ II II (Por)Fe + NO ⎯⎯ [(Por)Fe ||(NO)] → (Por)Fe (NO) ‡ (3) the understanding of the nitrosylation mechanism of met-Mb. The large and positive ∆V on (Table1) and ∆S‡ values indicated a limiting dissociative mechanism, controlled by the This is in agreementon with the fact that positive activation volumes for diffusion in lability of a water molecule, similar to the reactivity pattern observed for water-soluble Por various solvents are a result of the viscosity increase with ‡increasing pressure [35]. The systems. Attention was drawn to the fact that the value of ∆V on for met-Mb nitrosylation II dynamicsis significantly of NO largerbinding than to theFe correspondingporphyrins are value different for ferric-porphyrin than for CO binding. model Several systems. or- dersThis of observation magnitude waslower ascribed second-order to the structural rate constants reorganization for CO coordination, occurring in together the protein with thepocket negative upon Δ coordinatedV‡on, provide water confirmation release [30 ].that in this case, an activation-limited mecha- ‡ ‡ nism existsBesides (𝑘 myoglobin,≫𝑘). In particularthe activation-limited attention was mechanism also devoted (ΔV toon the = Δ reactivityV a), the negative of cy- ‡ II ΔVtochromeon value P450 is determined towards small by Fe molecules-L bond (O formation2, NO, and and CO). concomit Cytochromesant change P450, due in spin to their state. importantIt was surprising biological functions,to observe untypical similar NO active binding site with dynamics cysteine for thiolate pentacoordinate ligation to ferric- the monohydroxoiron center, and TMPS spin-state [27]. In equilibria this case, ofmuch the restinglower reaction state, are rates, subjects concomitant of intensive with research. negative ΔVDetailed‡on allowed mechanistic to conclude studies that on the P450 reactivity nitrosylation pattern played is not aalways crucial cont rolerolled in understanding by the lability ofthe the biologicaliron(III) center function (as principlewould be of expected this protein. in the High-pressure case of the diffus studiesion-limited on the reversible reactions), butNO can binding be determined by two forms by the of P450, spin namelydensity substrate-freereorganization (six-coordinate, and accompanying low-spin structural complex with water coordinated trans to Cys) and camphor-bound (five-coordinate, high-spin changes [27]. Comparison of typical volume profiles determined for six-coordinate diaqua complex), as well as the heme–thiolate porphyrin model (Figure 12A), also contributed to vs. five-coordinate monohydroxo iron(III) porphyrins are presented in Figure 10. this aspect [31,32]. Comparison of the results obtained for the model porphyrins with the ones for ferri- heme proteins was crucial to reveal the role of an amino acid chain surrounding the active center: polarity of the protein pocket, hydrogen-bonding interactions, prosthetic group solvent-accessibility, etc. The first high-pressure studies concerning reversible NO binding by heme protein were performed for met-myoglobin (metMb). A successful collaboration of three research groups (G. Stochel, R. van Eldik and P. Ford) resulted in a joint publication reporting high-pressure effects on the nitrosylation reaction rates [30]. The semi-independent results were performed by using two techniques: laser flash-photolysis

Molecules 2021, 26, x FOR PEER REVIEW 12 of 24

and stopped-flow. The application of these two fundamentally different techniques resulted in the determination of a set of activation parameters being in good agreement and providing insight into the understanding of the nitrosylation mechanism of met-Mb. The large and positive ΔV‡on (Table 1) and ΔS‡on values indicated a limiting dissociative mechanism, controlled by the lability of a water molecule, similar to the reactivity pattern observed for water-soluble Por systems. Attention was drawn to the fact that the value of ΔV‡on for met-Mb nitrosylation is significantly larger than the corresponding value for ferric-porphyrin model systems. This observation was ascribed to the structural reorganization occurring in the protein pocket upon coordinated water release [30]. Besides myoglobin, particular attention was also devoted to the reactivity of cytochrome P450 towards small molecules (O2, NO, and CO). Cytochromes P450, due to their important biological functions, untypical active site with cysteine thiolate ligation to the iron center, and spin-state equilibria of the resting state, are subjects of intensive research. Detailed mechanistic studies on P450 nitrosylation played a crucial role in understanding the biological function principle of this protein. High-pressure studies on the reversible NO binding by two forms of P450, namely substrate-free (six-coordinate, low-spin complex with water coordinated trans to Cys) and camphor-bound (five-coordinate, high-spin Molecules 2021, 26, 4947 12 of 24 complex), as well as the heme–thiolate porphyrin model (Figure 12A), also contributed to this aspect [31,32].

(A) (B)

− FigureFigure 12. 12. ((AA)) Synthetic Synthetic heme–thiolate heme–thiolate complex, complex, SR SR complex, (B) model ofof P450P450 bearingbearing R-SOR-SO3− asas − 3 an axial ligand (R-SO3 −). Reprinted with permission from Journal of the American Chemical Society an axial ligand (R-SO3 ). Reprinted with permission from Journal of the American Chemical Society 2005, 127, 5360–5375Copyright 2005 American Chemical Society and Journal of the American Chemical 2005, 127, 5360–5375 Copyright 2005 American Chemical Society and Journal of the American Chemical Society 2006, 128, 13611–13624Copyright 2006 American Chemical Society [32,33]. Society 2006, 128, 13611–13624 Copyright 2006 American Chemical Society [32,33].

TheThe activation activation volumes volumes determined determined for for NO NO binding binding to to these these two two P450 P450 forms forms revealed revealed differentdifferent nitrosylation nitrosylation mechanis mechanismsms [31]. [31]. For For substrate-free substrate-free cytochrome cytochrome P450 P450 a a mechanistic mechanistic scenarioscenario similar similar to the one forfor metMbmetMb waswas observed,observed, which which is is reflected reflected in in positive positive values values of ‡ ‡ ‡‡ ‡ of∆ VΔVononand and∆ ΔSSonon (Table1 1).). TheThe positivepositive ∆ΔV onon correspondscorresponds to to a a typical typical limiting limiting dissocia- dissocia- tivetive ligand-substitution ligand-substitution mechanism, mechanism, in in which which the the dissociation dissociation of of coordinated coordinated water water limits limits ‡‡ 33 thethe NO NO binding binding rate. SignificantlySignificantly largerlarger ∆ΔVV onon == +28 +28 cm cm /mol/mol in in comparison comparison to to metMb metMb representsrepresents a a much much higher higher structural structural rearrangem rearrangementent in in the the protein protein pocket pocket of of substrate-free substrate-free P450,P450, as as well well as as a aspin-state spin-state change change on on goin goingg from from the the low-spin low-spin six-coordinate six-coordinate complex complex to high-spinto high-spin five-coordinate five-coordinate transition-state transition-state intermediate intermediate [31]. [ 31The]. Themuch much smaller smaller positive pos- ‡ 3 ΔitiveV‡on ∆= V6 cmon 3=/mol 6 cm was/mol determined was determined for NO binding for NO to bindingsynthetic to heme–thi syntheticolate heme–thiolate six-coordi- natesix-coordinate complex (SR) complex in methanol (SR) in [32], methanol a model [32], of a P450 model cytochrome, of P450 cytochrome, allowing us allowing to estimate us to III theestimate participation the participation of factors of other factors than other FeIII than-H2O Fe bond-H 2breakageO bond breakage (spin-state (spin-state change and change re- organizationand reorganization in the protein in the protein pocket). pocket). ForFor camphor-bound camphor-bound P450P450 (P450(camph)),(P450(camph)), a a completely completely different different mechanistic mechanistic pathway path- waywas was observed observed that isthat expressed is expressed by negative by negative values values for both for activationboth activation volume volume (Table 1(Table) and ‡ 1)entropy and entropy determined determined for NO for binding. NO binding. Substantially Substantially negative negative∆V onΔVis‡on consistent is consistent with with the five-coordinate nature of P450(camph) form, for which the formation of [(Por)FeIII||(NO)] intermediate complex before NO bonding is expected [31]. Other studied valuable cytochrome P450 models were iron–porphyrin complexes, in − which the thiolate group was substituted by a R-SO3 ligand (Figure 12B). Depending on the solvent applied in the study, non-coordinating toluene vs. coordinating methanol, − the RSO3 complex exists as five-coordinate or six-coordinate species, respectively [33]. − Based on the activation parameters, it was clear that both the R-SO3 complex in toluene − (R-SO3 (toluene)) and five-coordinate P450(camph) follow the same mechanism, dominated by Fe-NO bond formation. However, a comparison of the activation volume profiles indicated differences in the transition states arising from dissimilar contributions from bond formation and spin state change (compare Figures 13B and 14A). Surprisingly, the − − reactivity profile of the six-coordinate R-SO3 complex in methanol (R-SO3 (MeOH)) differs from the six-coordinate substrate-free P450. High-pressure studies on the NO binding to the − ‡ R-SO3 (MeOH) complex revealed a significantly negative ∆V on value, indicating a volume collapse ongoing from the substrate to the transition state. An associative interchange − mechanism determined for the R-SO3 (MeOH) complex was related to the high spin nature of the complex, which is believed to govern the reactivity mechanism. This contrasts the low-spin six-coordinate P450 case, as the dissociative mechanism controls nitrosylation in such a system. The opposite effect is nicely illustrated by the volume profiles reported in Figures 13A and 14B. Molecules 2021, 26, x FOR PEER REVIEW 13 of 24 Molecules 2021, 26, x FOR PEER REVIEW 13 of 24

the five-coordinate nature of P450(camph) form, for which the formation of III [(Por)FeIII||(NO)] intermediate complex before NO bonding is expected [31]. Other studied valuable cytochrome P450 models were iron–porphyrin complexes, in which the thiolate group was substituted by a R-SO3− ligand (Figure 12B). Depending on which the thiolate group was substituted by a R-SO3− ligand (Figure 12B). Depending on the solvent applied in the study, non-coordinating toluene vs. coordinating methanol, the RSO3− complex exists as five-coordinate or six-coordinate species, respectively [33]. Based RSO3− complex exists as five-coordinate or six-coordinate species, respectively [33]. Based ̶ on the activation parameters, it was clear that both the R-SO3̶ complex in toluene (R-SO3− on the activation parameters, it was clear that both the R-SO3 complex in toluene (R-SO3− (toluene)) and five-coordinate P450(camph) follow the same mechanism, dominated by Fe- (toluene)) and five-coordinate P450(camph) follow the same mechanism, dominated by Fe- NO bond formation. However, a comparison of the activation volume profiles indicated differences in the transition states arising from dissimilar contributions from bond formation and spin state change (compare Figures 13B and 14A). Surprisingly, the reactivity profile of the six-coordinate R-SO3−complex in methanol (R-SO3−(MeOH)) differs from the six- profile of the six-coordinate R-SO3−complex in methanol (R-SO3−(MeOH)) differs from the six- coordinate substrate-free P450. High-pressure studies on the NO binding to the R- SO3−(MeOH) complex revealed a significantly negative ΔV‡on value, indicating a volume col- SO3−(MeOH) complex revealed a significantly negative ΔV‡on value, indicating a volume collapse ongoing from the substrate to the transition state. An associative interchange mechanism determined for the R-SO3−(MeOH) complex was related to the high spin nature of the anism determined for the R-SO3−(MeOH) complex was related to the high spin nature of the complex, which is believed to govern the reactivity mechanism. This contrasts the low- Molecules 2021, 26, 4947 spin six-coordinate P450 case, as the dissociative mechanism controls nitrosylation13 in of such 24 a system. The opposite effect is nicely illustrated by the volume profiles reported in Fig- ures 13A and 14B.

(A) (B) FigureFigure 13. 13.Volume Volume proﬁles profiles for for reversible reversible NO NO binding binding to to (A ()A substrate-free) substrate-free (P450) (P450) and and (B ()B camphor) camphor bound bound (P450(camph)) (P450(camph)) cytochrome.cytochrome.

(A) (B) − − Figure 14. Volume profiles for reversible NO binding to R-SO− 3− model in (A) toluene (R-SO− 3−(toluene)) and (B) in methanol FigureFigure 14. 14.Volume Volume proﬁles profiles for for reversible reversible NO NO binding binding to R-SO to R-SO3 model3 model in (Ain) ( tolueneA) toluene (R-SO (R-SO3 (toluene)3 (toluene))) and and (B (B) in) in methanol methanol (RSO3−(MeOH)). (RSO−3−(MeOH)). (RSO3 (MeOH)).

As was described above, activation volumes obtained from high-pressure kinetic studies for reversible NO binding to various iron porphyrin model systems, as well as heme

proteins, provide crucial information that leads to conclusions regarding the molecular mechanisms that govern the nitrosylation of heme centers. Volume profiles constructed based on the obtained data nicely present the differences in the position of transition states and allow graphically expressing the influence of various factors affecting it. The studies cited above demonstrate the invaluable impact of ∆V‡ in revealing the influence of the electronic nature of the porphyrin, the axial ligands present, the type of solvent (coordinating vs. non-coordinating), pH (influencing ionization state of protic groups), and amino acid chain that surrounds the active site on the ligand substitution behavior. Activation volumes are also a significant help in the identification of the relevant factors tuning the biological function of specific proteins.

4. Reactions of NO with Model FeII Complexes In the literature, we can ﬁnd examples of the application of high-pressure kinetic methods in mechanistic studies of the reactions of nitric oxide (NO) with iron(II) complexes, e.g., polyaminocarboxylates, cyanoferrates [36,37], or others [38,39]. The importance of mechanistic studies on such complexes is the comparison with water-exchange reactions that were clearly shown to control the nitrosylation process [37,40–44]. In this section, Molecules 2021, 26, 4947 14 of 24

we discuss a few important systems in which the high-pressure measurements helped to understand the nature of the underlying reaction mechanism. An interesting example of the application of high-pressure methods to improve our understanding of the underlying mechanism is the reaction known to every high school chemistry student, the so-called “brown-ring test” for nitrate [44]. The mentioned reaction is used as a “chemical test” that helps to demonstrate the presence of nitrate or nitrite ions in an aqueous solution. The test consists of the reduction of nitrate and nitrite to NO by an excess of Fe(II), which in the presence of concentrated sulfuric acid forms a characteristic brown-ring. Although the reaction is known for more than 100 years, the details of its mechanism were investigated relatively recently [45]. The process involves the formation of a characteristic green-brown colored ring at the junction of the two solutions layers. This brown-green color is provided by the formation of a Fe-NO complex in the reaction 2+ between [Fe(H2O)6] and NO (see Reaction (4)):

II 2+ II 2+ [Fe (H2O)6] + NO [Fe (H2O)5(NO)] + H2O (4)

2+ During the reaction, one water molecule in [Fe(H2O)6] is displaced rapidly by the 6 −1 −1 NO molecule (kon = 1.42 × 10 M s ). Detailed studies [44] showed that the product is formally stabilized as the FeIII-NO− complex in which electron density is shifted from the metal center to the ligand that results in the reduction of NO to NO− and formation of the 2+ iron(III)-nitrosyl complex. The back reaction involves the formation of [Fe(H2O)6] and −1 release of NO (koff = 3240 ± 750 s ) (see reaction 4). The effect of pressure on the described reaction was studied using the high-pressure ﬂash-photolysis technique in the pressure range 0.1–170 MPa (20 ◦C, pH = 5.0, 0.2 M acetate buffer) resulting in small positive volumes of activation (Table2): +6.1 ± 0.2 cm3 mol−1 and +1.3 ± 0.2 cm3 mol−1 for the “on” and “off” reactions, respectively. The activation parameters obtained from the applied measurements suggest that in both processes ligand substitution follows a dissociative interchange (Id) process [44]. Thus, it can be concluded that the forward reaction involves partial Fe-H2O bond breakage, prior to NO binding, whereas in the back reaction the Fe-NO bond partially dissociates and is followed by the coordination of H2O. This is shown in the volume proﬁle for the reaction presented in Figure 15.

Table 2. Summary of activation-volume data for the reversible coordination of NO by a series of FeII(L) complexes, where L represents a series of polyaminocarboxylate complexes.

∆V‡ ∆V‡ Complex on off Reference (cm3/mol) (cm3/mol) II 2+ [Fe (H2O)6] +6.1 ± 0.4 +1.3 ± 0.2 [44] II 2+ [Fe (edta)(H2O)] +4.1 ± 0.2 +7.6 ± 0.6 [37] II 2+ [Fe (hedtra)(H2O)] +2.8 ± 0.1 +4.4 ± 0.8 [37] II 2+ [Fe (nta)(H2O)2] −1.5 ± 0.1 −3.5 ± 0.7 [37] II 2+ [Fe (mida)(H2O)3] +7.6 ± 0.4 +6.8 ± 0.4 [37] II 2+ [Fe (mida)2(H2O)] +8.1 ± 0.2 +5.1 ± 0.5 [37] II 3− [Fe (CN)5(H2O)] +17.4 ± 0.3 +7.1 ± 0.2 [39]

2+ Mechanistic studies on the water-exchange reaction in [Fe(H2O)6] were performed using HP 17O-NMR techniques as described in Section2 of this review. The water-exchange reaction has a rate constant of 4.4 × 106 s−1 at 20 ◦C and the volume of activation for this reaction was reported to be +3.8 ± 0.2 cm3 mol−1, which suggests that it also follows a dissociative interchange (Id) mechanism [40]. This means that both the nitrosylation and water-exchange reactions occur according to the same Id mechanism for which the water- exchange process controls both the rate and the nature of the NO binding mechanism. In a 2+ recent publication by Klüfers and Monsch [45], the crystal structure of [Fe(H2O)5(NO)] Molecules 2021, 26, x FOR PEER REVIEW 15 of 24

ΔV‡on luhan ΔV‡off luhan Complex Reference (cm3 /mol) (cm3 /mol) [FeII(H2O)6]2+ +6.1 ± 0.4 +1.3 ± 0.2 [44] [FeII(edta)(H2O)]2+ +4.1 ± 0.2 +7.6 ± 0.6 [37]

II 2 2+ Molecules 2021, 26, 4947 [Fe (hedtra)(H O)] +2.8 ± 0.1 +4.4 ± 0.8 [37] 15 of 24 [FeII(nta)(H2O)2]2+ −1.5 ± 0.1 −3.5 ± 0.7 [37] [FeII(mida)(H2O)3]2+ +7.6 ± 0.4 +6.8 ± 0.4 [37] [FeII(mida)2(H2O)]2+ +8.1 ± 0.2 +5.1 ± 0.5 [37] ◦ was resolved,II and3− the Fe-N-O had a bond angle of 168 . This reopened the discussion on [Fe (CN)5(H2O)] +17.4 ± 0.3 +7.1 ± 0.2 [39] the nature of the Fe-NO complex, whether it is Fe(I)-NO+, Fe(II)-NO•, or Fe(III)-NO− [45].

(A) (B)

2+2+ FigureFigure 15. 15. (A(A) )Volume Volume profile proﬁle for for the the reaction reaction of [Fe(H2O)O)66]] withwith NO. NO. (B (B) A) A picture picture of of the the brown-ring brown-ring formed formed during during the the testtest for for nitrate nitrate or or nitrite nitrite [45]. [45]. Copyright Copyright Wiley-VCH Wiley-VCH GmbH. GmbH. Reproduced Reproduced with with permission. permission.

MechanisticAnother interesting studies on system the water-exchange in which the application reaction in of [Fe(H high-pressure2O)6]2+ were methods performed was usinginvaluable HP 17 forO-NMR the investigation techniques ofas reactiondescribed mechanisms in Section is2 theof reactionthis review. of NO The with water-ex- a group changeof polyaminocarboxylate reaction has a rate complexesconstant of of 4.4 Fe ×II 10(edta,6 s−1 at hedtra, 20 °C and nta, andthe volume mida; see of Figureactivation 16)[ for37]. thisThe reaction performed was studies,reported including to be +3.8 an± 0.2 investigation cm3 mol−1, which of the suggests coordinated that it water-exchange also follows a dissociativeprocess [42 ,interchange46], allowed (I usd) mechanism not only to [40]. recognize This means the sequence that both of the the nitrosylation reaction but and also water-exchangegave us crucial reactions answers foroccur questions according concerning to the same the Id mechanistic mechanism naturefor which of thethe studiedwater- exchangecomplexes. process In general, controls polyaminocarboxylate both the rate and the complexes nature of of the iron(II) NO binding are known mechanism. to be highly In aeffective recent publication NO scavengers by Klüfers due and to their Monsch very [45], efﬁcient the crystal coordination structure of of NO [Fe(H [472O),485].(NO)] Since2+ wasit is resolved, known that and polyaminocarboxylatesthe Fe-N-O had a bond possessangle of the168°. desired This reopened pharmacokinetic the discussion property, on thetheir nature iron(II) of the complexes Fe-NO complex, were considered whether it as is reagentsFe(I)-NO+ that, Fe(II)-NO control•, theor Fe(III)-NO NO level− in[45]. the Molecules 2021, 26, x FOR PEER REVIEW 16 of 24 bloodstreamAnother interesting [49,50]. On system the other in hand,which they the haveapplication been used of high-pressure for the removal methods of NO fromwas invaluableexhaust gasses, for the since investigation they can enhanceof reaction the mechanisms solubility of is NO thein reaction aqueous of solutions.NO with a group of polyaminocarboxylate complexes of FeII (edta, hedtra, nta, and mida; see Figure 16) [37]. The performed studies, including an investigation of the coordinated water-exchange process [42,46], allowed us not only to recognize the sequence of the reaction but also gave us crucial answers for questions concerning the mechanistic nature of the studied complexes. In general, polyaminocarboxylate complexes of iron(II) are known to be highly effective NO scavengers due to their very efficient coordination of NO [47,48]. Since it is known that polyaminocarboxylates possess the desired pharmacokinetic property, their iron(II) complexes were considered as reagents that control the NO level in the bloodstream [49,50]. On the other hand, they have been used for the removal of NO from exhaust gasses, since they can enhance the solubility of NO in aqueous solutions.

FigureFigure 16. 16. StructuresStructures of the the ligands: ligands: (A (A) edta) edta (ethylenediaminetetraacetate), (ethylenediaminetetraacetate), (B) ( Bhedtra) hedtra (hydroxyeth- (hydrox- ylenediaminetriacetate), (C) nta (nitrilotriacetate), and (D) mida (methyliminodiacetate). yethylenediaminetriacetate), (C) nta (nitrilotriacetate), and (D) mida (methyliminodiacetate).

TheThe studied studied complexes complexes are are seven-coordinate seven-coordinate species species with with one one water water molecule molecule that that is is boundbound to to thethe ironiron centercenter (for edta, hedtra, hedtra, and and mida) mida) or or six-coordinate six-coordinate species species with with two two co- coordinatedordinated water water molecules molecules (for (for nta). nta). It Itis isalso also worth worth mentioning mentioning that that the the polyaminocarbox- polyaminocar- ylate complexes of Fe(II) are extremely oxygen-sensitive; in the presence of O2 they form Fe(III) species that do not interact with NO at all. The reaction between the group of complexes ([FeII(L)(H2O)n] and NO, can be expressed by Reaction (5) (see below) and lead to the formation of stable adducts of FeII(L)(NO), where L = edta, hedtra, nta, and mida [37].

[Fe (L)(HO)]+NO ⇄ [Fe (L)(HO)(NO)] + HO (5) Interestingly, similar to that described in the previous example, the nitrosyl products are stabilized as FeIII-NO− complexes, due to a shift in charge density from the metal center to the NO ligand. It is worth noting that the observed nitrosylation reactions are very efficient with rate constants that vary from 1.6 × 106 M−1 s−1 for the (mida)2 complex to even 2.4 × 108 M−1 s−1 for the FeII(edta) complex! The rate constants for the nitrosylation reaction of the complexes increases in following order: edta > hedtra > nta > mida > (mida)2. The back reactions leading to NO release and formation of [FeII(L)(H2O)n] complexes (see reaction 5) are within the range from 0.11 to 3.2 × 103 s−1 [37]. For this group of compounds, the high-pressure stopped-flow technique was applied to study the pressure effect and allowed to obtain the results listed in Table 2. What is worth to underline for all complexes except for nta, the activation volumes reached small positive values that suggest the dissociative interchange (Id) mechanism for the nitrosylation reaction. Thus, the most probable sequence during the reaction is again the partial Fe- H2O bond breakage, followed by NO binding as was also described above for the [Fe(H2O)6]2+ complex [37]. Only in the case of the nta complex, negative activation volumes were found, indicating an associative interchange (Ia) mechanism for the nitrosylation reaction and suggesting that the reaction involves partial Fe-NO bond formation prior to the release of H2O. This perfectly agrees with the six-coordinate nature of the nta complex compared to the seven-coordinate nature of the other complexes as shown in Figure 17 and the results in Table 2.

Molecules 2021, 26, 4947 16 of 24

boxylate complexes of Fe(II) are extremely oxygen-sensitive; in the presence of O2 they form Fe(III) species that do not interact with NO at all. The reaction between the group II of complexes ([Fe (L)(H2O)n] and NO, can be expressed by Reaction (5) (see below) and lead to the formation of stable adducts of FeII(L)(NO), where L = edta, hedtra, nta, and mida [37]. II II [Fe (L)(H2O)n] +NO [Fe (L)(H2O)n−1(NO)] + H2O (5) Interestingly, similar to that described in the previous example, the nitrosyl products are stabilized as FeIII-NO− complexes, due to a shift in charge density from the metal center to the NO ligand. It is worth noting that the observed nitrosylation reactions are very 6 −1 −1 efﬁcient with rate constants that vary from 1.6 × 10 M s for the (mida)2 complex to even 2.4 × 108 M−1 s−1 for the FeII(edta) complex! The rate constants for the nitrosylation reaction of the complexes increases in following order: edta > hedtra > nta > mida > (mida)2. II The back reactions leading to NO release and formation of [Fe (L)(H2O)n] complexes (see reaction 5) are within the range from 0.11 to 3.2 × 103 s−1 [37]. For this group of compounds, the high-pressure stopped-ﬂow technique was applied to study the pressure effect and allowed to obtain the results listed in Table2. What is worth to underline for all complexes except for nta, the activation volumes reached small positive values that suggest the dissociative interchange (Id) mechanism for the nitrosylation reaction. Thus, the most probable sequence during the reaction is again the partial Fe-H2O bond breakage, followed by NO binding as was also described above for 2+ the [Fe(H2O)6] complex [37]. Only in the case of the nta complex, negative activation volumes were found, indicating an associative interchange (Ia) mechanism for the nitrosylation reaction and suggesting Molecules 2021, 26, x FOR PEER REVIEWthat the reaction involves partial Fe-NO bond formation prior to the release of H2O.17 of This 24 perfectly agrees with the six-coordinate nature of the nta complex compared to the seven- coordinate nature of the other complexes as shown in Figure 17 and the results in Table2.

(A) (B)

Figure 17. Volume profiles for the reactions of (A) seven-coordinate [FeIIII(edta)(H2O)]2+2+ and (B) six-coordinate Figure 17. Volume proﬁles for the reactions of (A) seven-coordinate [Fe (edta)(H2O)] and (B) six-coordinate [FeIIII(nta)(H2O)2]2+ 2+complexes with NO [37]. [Fe (nta)(H2O)2] complexes with NO [37].

AdditionalAdditional 1717O-NMRO-NMR studies studies of of the the water-exchange water-exchange reaction reaction for for this this group group of of com- com- plexesplexes demonstrated demonstrated that that the the water-exchange water-exchange pr processocess controls controls the the substitution substitution of of NO NO by by Fe(II)Fe(II) polyaminocarboxylate polyaminocarboxylate complexescomplexes [37[37,42,43,46].,42,43,46]. On On the the other other hand, hand, it wasit was shown shown that thatthe chelatethe chelate ligand ligand controls controls the rate the of rate the of water-exchange the water-exchange process, process, but also but has also an inﬂuence has an influenceon the nature on the of nature the underlying of the underlying mechanism mechanism [42,43,46 ].[42,43,46]. TheThe difference difference in in the the nitrosylation nitrosylation mechanism mechanism for for the the nta ntacomplex complex was was accounted accounted for asfor a result as a result of its ofsix-coordinate its six-coordinate nature nature in solution in solution and 18 and valence 18 valence electron electron character, character, com- paredcompared to the to other the other complexes complexes that that are areseven- seven-coordinatecoordinate and and possess possess 20 20 valence valence electron electron character. The observed volume changes are related to exchanging H2O for the NO ligand in the complexes (seven- or six-coordinate) and likely changes in the formal oxidation state of the metal center and NO [37]. It is noteworthy to mention that similar activation volume values were found for the forward and backward reactions, i.e., positive activation volumes were found for edta, hedtra, mida, and (mida)2 complexes, and slightly negative values for the nta complex (Table 2). These values clearly indicate that the backward reactions for edta, hedtra, mida and (mida)2 complexes also follow a dissociative interchange (Id) mechanism. The FeII(nta)(NO) system is an exception with an Ia mechanism for both the “on” and “off” reactions [37]. The final example we would like to discuss concerns the formation and dissociation reactions of [FeII(CN)5(NO)]3−, the complex that is formed during the interaction between NO and [FeII(CN)5(H2O)]3− (see Reaction (6)) [39]. Especially, an interesting aspect of the system seems to be the characterization of the NO release reaction, since the similar iron(III) complex is referred to as nitroprusside ([Fe(CN)5(NO)]2−), which for decades is well-known as NO-releasing agent in biological medium and is used for clinical purposes [38].

[Fe (CN)(HO)] +NO ⇄ [Fe (CN)(NO)] +HO (6)

The process of [Fe(CN)5(NO)]3− formation was found to be quite efficient with kon = 250 ± 10 M−1 s−1 (25.4 °C, I = 0.1 M, pH 7.0), and the back reaction expressed by rate constant koff = (1.58 ± 0.06) × 10−5 s−1 (25.0 °C, I = 0.1 M, pH 10.2) [39]. The activation volumes for both forward and backward reactions were found to be positive: +17.4 ± 0.3 cm3 mol−1 and +7.1 ± 0.2 cm3 mol−1 for “on” and “off” reactions, respectively. In the case of the forward reaction, the activation volume value suggests the operation of a limiting dissociative mechanism (D). Thus the rate-controlling process is the dissociation of coordinated H2O to form a five-coordinate complex, followed by a fast reaction

Molecules 2021, 26, 4947 17 of 24

character. The observed volume changes are related to exchanging H2O for the NO ligand in the complexes (seven- or six-coordinate) and likely changes in the formal oxidation state of the metal center and NO [37]. It is noteworthy to mention that similar activation volume values were found for the forward and backward reactions, i.e., positive activation volumes were found for edta, hedtra, mida, and (mida)2 complexes, and slightly negative values for the nta complex (Table2). These values clearly indicate that the backward reactions for edta, hedtra, mida and (mida)2 complexes also follow a dissociative interchange (Id) mechanism. The II Fe (nta)(NO) system is an exception with an Ia mechanism for both the “on” and “off” reactions [37]. The ﬁnal example we would like to discuss concerns the formation and dissociation II 3− reactions of [Fe (CN)5(NO)] , the complex that is formed during the interaction between II 3− NO and [Fe (CN)5(H2O)] (see Reaction (6)) [39]. Especially, an interesting aspect of the system seems to be the characterization of the NO release reaction, since the similar iron(III) 2− complex is referred to as nitroprusside ([Fe(CN)5(NO)] ), which for decades is well-known as NO-releasing agent in biological medium and is used for clinical purposes [38].

II 3− II 3− [Fe (CN)5(H2O)] + NO [Fe (CN)5(NO)] + H2O (6)

3− The process of [Fe(CN)5(NO)] formation was found to be quite efﬁcient with −1 −1 ◦ kon = 250 ± 10 M s (25.4 C, I = 0.1 M, pH 7.0), and the back reaction expressed −5 −1 ◦ by rate constant koff = (1.58 ± 0.06) × 10 s (25.0 C, I = 0.1 M, pH 10.2) [39]. The activation volumes for both forward and backward reactions were found to be positive: +17.4 ± 0.3 cm3 mol−1 and +7.1 ± 0.2 cm3 mol−1 for “on” and “off” reactions, respectively. In the case of the forward reaction, the activation volume value suggests Molecules 2021, 26, x FOR PEER REVIEW 18 of 24 the operation of a limiting dissociative mechanism (D). Thus the rate-controlling process is the dissociation of coordinated H2O to form a ﬁve-coordinate complex, followed by a fast reaction with NO. For the back reaction, the dissociative mechanism (D) is also withfavored, NO. which For the means back reaction, the reaction the dissociativ involves Fe-NOe mechanism bond cleavage (D) is also prior favored, to the which coordination means theof H reaction2O (see involves Figure 18 Fe-NO). However, bond clea it shouldvage prior be mentionedto the coordination that NO of release H2O (see could Figure be due18). However,to an alternative it should decomposition be mentioned that route NO that release involves could the be due decay to an of alternative a tetracyanonitrosyl decompo- sitioncomplex route [39 that]. involves the decay of a tetracyanonitrosyl complex [39].

II 3− Figure 18. VolumeVolume proﬁleprofile forfor the the reversible reversible coordination coordination of of NO NO to to [Fe [Fe(CN)II(CN)5H5H2O]2O]3− according to Reaction (6) [[39]39]..

Finally, it must be mentioned that several studies in which the water-exchangewater-exchange process for a variety variety of of iron(II) iron(II) and and iron(III) iron(III) complexe complexes,s, can can be be found found in inthe the literature literature [40–43,46]. [40–43,46 In]. theseIn these studies, studies, high-pressure high-pressure methods methods were were employed employed to assist to assist the theunderstanding understanding of the of chemistrythe chemistry of the of complexes, the complexes, the nature the nature of their of theirinteraction, interaction, the influence the inﬂuence of the ofmetal the center metal [42],center and [42 the], and effect the of effect the chelate of the chelateligand [41,43] ligand on [41 the,43 ]water-exchange on the water-exchange process. process.

5. NO Binding to Cobalamin and Cobalt Porphyrins Biological aspects of nitric oxide interactions with metal ions are primarily associated with the activation of metalloenzymes (such as guanylyl cyclase) or their inhibition (observed for cytochrome oxidase or catalase). Interest regarding the reaction mechanisms involving NO has been mostly focused on heme iron complexes and their model systems. Slightly less effort has so far been devoted to cobalt complexes, which have attracted attention due to the known biological effects of the interaction between NO and nitrile hydratase, an enzyme that contains ions of both the aforementioned metals in its active center [51,52]. Studies of cobalt complexes nitrosylation were, therefore, often carried out referring to the better-known iron-based systems. Elucidation of the differences between the reactivity of the two metals towards NO was performed using model porphyrin complexes [53,54]. An expected conclusion from the early studies of this type was the dependence of the reaction course on the oxidation state of the metal and the conditions, pH in particular. Findings by Laverman and Ford, who treated CoII(TPPS) to some extent as a reference for iron porphyrin complexes, showed closer similarity between Co(II) and Fe(II) complexes than between Fe(II) and Fe(III) in terms of the reactivity towards NO. The reactions were studied at pH = 7, using high-pressure techniques. The obtained ΔV# values indicate that the reactions outlined in (7)

k M(Por) +NO ⇄ M(Por)(NO) (7) k

follow D and ID mechanisms for ligand exchange on M(III) and M(II), respectively [26]. In a similar context, Roncaroli et al. reported comprehensive studies on model Co(III) porphyrins [55]. In contrast to MII(Por)(NO+) species previously postulated as a reaction product of the reactions involving Fe(III) porphyrins, indisputable spectroscopic evidence

Molecules 2021, 26, 4947 18 of 24

5. NO Binding to Cobalamin and Cobalt Porphyrins Biological aspects of nitric oxide interactions with metal ions are primarily associated with the activation of metalloenzymes (such as guanylyl cyclase) or their inhibition (observed for cytochrome oxidase or catalase). Interest regarding the reaction mechanisms involving NO has been mostly focused on heme iron complexes and their model systems. Slightly less effort has so far been devoted to cobalt complexes, which have attracted attention due to the known biological effects of the interaction between NO and nitrile hydratase, an enzyme that contains ions of both the aforementioned metals in its active center [51,52]. Studies of cobalt complexes nitrosylation were, therefore, often carried out referring to the better-known iron-based systems. Elucidation of the differences between the reactivity of the two metals towards NO was performed using model porphyrin complexes [53,54]. An expected conclusion from the early studies of this type was the dependence of the reaction course on the oxidation state of the metal and the conditions, pH in particular. Findings by Laverman and Ford, who treated CoII(TPPS) to some extent as a reference for iron porphyrin complexes, showed closer similarity between Co(II) and Fe(II) complexes than between Fe(II) and Fe(III) in terms of the reactivity towards NO. The reactions were studied at pH = 7, using high-pressure techniques. The obtained ∆V# values indicate that the reactions outlined in (7)

kon M(Por) + NO M(Por)(NO) (7) koff

follow D and ID mechanisms for ligand exchange on M(III) and M(II), respectively [26]. In a similar context, Roncaroli et al. reported comprehensive studies on model Co(III) porphyrins [55]. In contrast to MII(Por)(NO+) species previously postulated as a reaction product of the reactions involving Fe(III) porphyrins, indisputable spectroscopic evidence clearly shows that MIII(Por)(NO−) is formed in the case of Co(III) reactions with NO according to the following reaction sequence:

III III − − Co (Por) + 2NO → Co (Por)(NO ) + NO2 (8)

It was observed that the details of the reaction mechanism depended on the reaction medium. To provide in-depth insight into the factors contributing to these differences, the reaction course was followed over a wide range of pH. As the pH was lowered below 3, the characteristics of the kinetic traces changed from biphasic to monophasic, suggesting a single reaction step. Based on the kinetic data, it was concluded that product formation under strongly acidic conditions is preceded by a rate-limiting step of water exchange to form III [Co (Por)(NO)(H2O)]. The latter species immediately reacts with the second NO molecule. III III − The resulting [Co (Por)(NO)2] intermediate is instantly converted to [Co (NO )] by an inner-sphere electron transfer. Reasons for the greater complexity of the mechanism carried out under milder conditions (pH > 3) were perceived in the possible participation of nitrite − impurities that undergo protonation in more acidic solutions. The competition of NO2 and NO in the formation of the first intermediate justifies the more complex form of the rate law: − kobs = kNO[NO] + k − NO (9) NO2 2 In contrast to iron porphyrins, which show relatively high stability of a [FeII(Por)(NO+)] intermediate, its cobalt analog, namely [CoIII(Por)(NO)], is highly prone to further trans- − formations. The formation of a subsequent intermediate, [Co(Por)(NO2 )(NO)], which slowly decomposes to the final product, was found based on the determined rate constant − ‡ dependencies on both NO and NO2 concentrations, supported by a positive ∆V value of 3 +13 cm /mol for Co(TPPS)(H2O) at pH = 1. Thus, the mechanism of reductive nitrosylation of cobalt porphyrins differs significantly from the mechanisms of analogous reactions involving iron complexes. Molecules 2021, 26, 4947 19 of 24

In the human body, cobalt(III) complexes are represented mainly by cofactors of vitamin B12. Despite the common macrocyclic framework with porphyrins, the corrin system exerts a peculiar influence on the properties of the metal center, which is of interest, among others, because of the potential activation of the small molecules. Therefore, along with porphyrins, cobalamins (Cbl) represent an attractive and biologically important object of study, for which the application of high-pressure kinetic techniques has contributed to the elucidation of the NO reactions’ mechanisms. Among the naturally occurring vitamin B12 derivatives, aquacobalamin (Cbl(H2O)) has attracted particular attention because of its substantial ability to substitute the water molecule. Of all the six-coordinated Co(III)-based forms, only one gave rise to a discussion of potential NO binding under physiological conditions [56,57]. However, this hypothesis was negatively verified, and the observed spectroscopic features were attributed solely to the presence of nitrite impurities [58,59]. Under more acidic conditions, Cbl(H2O) reacts with NO to give a nitrosyl derivative [60]. Surprisingly, the final product is CblII(NO), or, given the actual electron density distribution, CblIII(NO−), indicating that reductive nitrosylation has occurred. Comparison with previously described porphyrins revealed several similarities but also some differences in the details of these reaction mechanisms. Their convergence is manifested, among others, by the biphasic nature of the reaction occurring in the presence of HNO2. The product of − the first step, Cbl(NO2 ), is formed at a rate virtually independent of NO concentration Molecules 2021, 26, x FOR PEER REVIEW and very similar to that observed in the presence of pure HNO2 [59]. This finding suggests 20 of 24 to rule out the possibility of a direct reaction of Cbl(H2O) with NO, regardless of the conditions. At a pH < 4, the nitrite mediated reaction proceeds to form the final product, but the determined concentration dependencies, with respect to both NO and NO2, gave rather unexpected courses. While the reaction rate decreases with increasing NO concentration, it overall course of the reductive nitrosylation of Cbl(H2O) is given by the following equa- increases with the square of [HNO2]. Moreover, the reaction yield drops with increasing tion: concentrations of both species. This clearly indicates that the [HNO2]-dependent reverse reaction controls the rate of formation of the final product. Hence, the overall course of the reductive nitrosylationCbl of Cbl(H(NO2O)) +2NO+ is given by H theO following ⇄ Cbl equation:(NO ) +2HNO (10)

− − The role of nitriteCbl under(NO2 ) acidic + 2NO +conditionsH2O Cbl (wasNO )clarified + 2HNO2 by considering(10) the properties

of the dihydrobenzimidazoleThe role of nitrite under acidic group conditions (DMBI), was clariﬁed which, by in considering the natural the properties “base ofon” form, coor- dinatesthe to dihydrobenzimidazole the cobalt ion on group the (DMBI), opposite which, side in the of natural water “base in on”Cbl(H form,2 coordinatesO). The substitution of H2O forto theNO cobalt2− has ion been on the shown opposite to side markedly of water inshift Cbl(H the2O). p TheKa value substitution of DMBI, of H2O which, in very − acidic forsolutions, NO2 has allows been shown dechelation to markedly (“base shift off” the p Kformation)a value of DMBI, and which,NO binding. in very The opposite acidic solutions, allows dechelation (“base off” formation) and NO binding. The opposite sequencesequence of events of events cannot cannot be realized. realized. The The detailed detailed mechanism mechanism of the multi-step of the reaction multi-step reaction of Cbl(Hof Cbl(H2O) with2O) with NO NO in in the the presencepresence of HNOof HNO2 at low2 at pH low is shown pH is in shown the following in the scheme following scheme (see Figure(see Figure 19): 19 ):

Figure 19. Reaction scheme for the reductive nitrosylation of Cbl(H2O) at low pH (according to [17]). Figure 19. Reaction scheme for the reductive nitrosylation of Cbl(H2O) at low pH (according to [17]). The instability of the CoIII-NO species, demonstrated previously for Co(III) por- Thephyrins, instability accounts of for the the practicalCoIII-NO inability species, of Cbl(H demonstrated2O) to react directly previously with NO. for Thus, Co(III) porphy- the mechanism of reductive nitrosylation of Cbl(H2O) is signiﬁcantly different from that of rins, accountsan analogous for reaction the practical involving inability Fe(III) porphyrins. of Cbl(H2O) to react directly with NO. Thus, the mechanism of reductive nitrosylation of Cbl(H2O) is significantly different from that of an analogous reaction involving Fe(III) porphyrins. While the issue of the potential interaction of Cbl(H2O) with NO has remained con- troversial for many years, such a reaction with Cbl(II) is virtually indisputable. It is supported by unequivocal evidence provided by a variety of spectroscopic techniques (UV– Vis, 1H-, 15N-, and 31P NMR). The results of kinetic investigations indicate a very high equilibrium constant (108 M−1 at pH = 7.0) for the following reaction: Cbl(II) + NO ⇄ Cbl(NO) (11) Based on NMR studies, the actual form of the final product was defined as Cbl(III)(NO−), in agreement with the products of reductive nitrosylation of Cbl(H2O) and Co(III) porphyrins. The activation parameters were determined for both the formation and decay of the nitrosyl complex by using laser flash photolysis and stopped-flow techniques, respectively. The effect of pressure was studied for both reactions. The small positive value of ΔV‡on for Cbl(III)(NO−) formation (+5.4 cm3/mol at pH = 7.4) suggested im- plementation of the dissociative interchange mechanism, which was possible due to the appearance of a water-bound intermediate. A similar type of mechanism is being realized in the reverse reaction forced by the use of NO-trapping technique, as indicated by the value of ΔV‡off = +7.9 cm3/mol. The volume profile for the reaction 11 is shown in Figure 20.

Molecules 2021, 26, 4947 20 of 24

While the issue of the potential interaction of Cbl(H2O) with NO has remained contro- versial for many years, such a reaction with Cbl(II) is virtually indisputable. It is supported by unequivocal evidence provided by a variety of spectroscopic techniques (UV–Vis, 1H-, 15N-, and 31P NMR). The results of kinetic investigations indicate a very high equilibrium constant (108 M−1 at pH = 7.0) for the following reaction:

Cbl(II) + NO Cbl(NO) (11)

Based on NMR studies, the actual form of the final product was defined as Cbl(III)(NO−), in agreement with the products of reductive nitrosylation of Cbl(H2O) and Co(III) porphyrins. The activation parameters were determined for both the formation and decay of the nitrosyl complex by using laser flash photolysis and stopped-flow techniques, respectively. The effect of pressure was studied for both reactions. The small positive value ‡ − 3 of ∆V on for Cbl(III)(NO ) formation (+5.4 cm /mol at pH = 7.4) suggested implementa- tion of the dissociative interchange mechanism, which was possible due to the appearance of a water-bound intermediate. A similar type of mechanism is being realized in the re- MoleculesMolecules 20212021,, 2626,, xx FORFOR PEERPEER REVIEWREVIEW 2121 ofof 2424 verse reaction forced by the use of NO-trapping technique, as indicated by the value of ‡ 3 ∆V off = +7.9 cm /mol. The volume profile for the reaction 11 is shown in Figure 20.

FigureFigure 20. 20. TheThe volume volume profile proﬁleprofile for for thethe reactionreaction ofof Cbl(II)Cbl(II) withwith NONO inin aqueousaqueous solutionsolution atat pHpH == 7.4.7.4.

OneOne of of the the biologicallybiologically relevant relevant pathwayspathways forfor thethe interactioninteraction ofof Cbl(HCbl(HCbl(H222O)O) with with NONO involvesinvolvesinvolves reactions reactionsreactions with withwith endogenous endogenous S-nitros S-nitrosothiolsS-nitrosothiolsothiols (RSNO). TheirTheir bindingbinding toto the the metalmetal center,center,center, regardless regardless ofof whetherwhether realizedrealized throughthrough aa sulfursulfur oror nitrogennitrogen atom,atom, usuallyusually leadsleads toto homolytichomolytic cleavage cleavage of of the the S-NO S-NO bond,bond, whichwhich maymay affectaffect nitricnitricnitric oxideoxideoxide homeostasis.homeostasis.homeostasis. ThisThis promptedprompted anan in-depthin-depth studystudy onon the the mechanisms mechanisms ofof these these interactions, interactions, consideringconsidering variousvarious potentialpotential reactionreaction sequencessequences sequences leadingleading leading toto to thethe the formationformation formation andand and furtherfurther further fatefate fateofof Cbl(RSNO).Cbl(RSNO). of Cbl(RSNO). Ap-Ap- plicationApplicationplication ofof high-pressure ofhigh-pressure high-pressure techniquestechniques techniques significantlysignificantly signiﬁcantly contributedcontributed contributed toto to thethe the elucidationelucidation of of the the mechanism of the reaction of glutathionylcobalamin (CblSR) with NO. The negative value mechanismmechanism ofof thethe reactionreaction ofof glutathionylcobalaminglutathionylcobalamin (CblSR)(CblSR) withwith NO.NO. TheThe negativenegative valuevalue of ∆V‡ (−9.6 cm3/mol) indicates an associative reaction pathway, leading to the formation ofof ΔΔVV‡‡ ((−−9.69.6 cmcm33/mol)/mol) indicatesindicates anan associativeassociative reactionreaction pathway,pathway, leadingleading toto thethe formationformation of S-nitrosoglutathionylcobalamin. In the following rapid step, it decomposes to GSNO ofof S-nitrosoglutathionylcobalamin.S-nitrosoglutathionylcobalamin. InIn thethe followingfollowing rapidrapid step,step, itit decomposesdecomposes toto GSNOGSNO and the reduced cobalt complex [61]: andand thethe reducedreduced cobaltcobalt complexcomplex [61]:[61]: NONO CblCbl − − SR SR + + NO NO ⇄ ⇄CblCbl − − S S ⇄⇄ Cbl Cbl((IIII))+RSNO+RSNO RR (12)(12) A similar mechanistic scenario has been observed for nitrosylation of thiolato ligand AA similar similar mechanistic scenario has been observed for nitrosylation of thiolato ligand ininin the thethe model model Fe(III)–thiolate Fe(III)–thiolate porphyrin porphyrinporphyrin complex. complex.complex. In InIn this thisthis case, case, high-pressure high-pressure kinetic kinetickinetic studies studiesstudies alsoalso revealedrevealed negativenegative ΔΔVV‡‡on‡on valuesvalues viz.viz. −−16.316.3 cmcm33/mol/mol3 pointingpointing towardstowards aa commoncommon asso-asso- also revealed negative ∆V on values viz. −16.3 cm /mol pointing towards a common ciativelyassociativelyciatively activatedactivated activated reactionreaction reaction pathwaypathway pathway [32].[32]. [ 32]. InInIn recent recent years, years, another another Reactive Reactive Nitrogen Nitrogen and and Oxygen Oxygen SpeciesSpecies (RNOS),(RNOS), namelynamely HNO,HNO, has has been been receiving receiving increasing increasing attention, attention, due due to to the the finding ﬁndingfinding of of itsits unique—and,unique—and, inin somesomesome cases, cases, eveneven complementarycomplementary complementary toto to NO—functionNO—function NO—function inin in biologicalbiological biological systemssystems systems [62].[62]. [62 In].In Inorderorder order toto thoroughlythoroughly exploreexplore thethe mechanismmechanism ofof thethe reactionreaction ofof HNOHNO withwith Cbl(HCbl(H22O),O), kinetickinetic studiesstudies werewere performedperformed overover aa widewide pHpH rangerange usingusing PiloPiloty’sty’s acidacid (PA)(PA) asas thethe nitroxylnitroxyl donordonor [63].[63]. SignificantSignificant differencesdifferences regarding,regarding, amongamong others,others, thethe rate-determiningrate-determining stepstep ofof Cbl(III)(NOCbl(III)(NO−−)) formationformation werewere foundfound dependingdepending onon thethe PAPA concentration.concentration. TheThe resultsresults ofof thethe kinetickinetic studiesstudies revealedrevealed thethe pKpKaa ofof nitroxylnitroxyl toto bebe 11.4.11.4. FollowingFollowing thethe pHpH scale,scale, partic-partic- ularlyularly interestinginteresting observationsobservations werewere mademade inin thethe neutralneutral range,range, wherewhere thethe reactionreaction prod-prod- uctuct isis formedformed remarkablyremarkably fastfast despitedespite thethe highhigh stabilitystability ofof PA.PA. ThisThis unusualunusual behaviorbehavior waswas explainedexplained onon thethe basisbasis ofof thethe formationformation ofof thethe Cbl(III)(PA)Cbl(III)(PA) intermediate,intermediate, whichwhich decaysdecays toto nitrosylcobalaminnitrosylcobalamin uponupon deprodeprotonationtonation andand S–NS–N bondbond breabreaking.king. High-pressureHigh-pressure tech-tech- niquesniques havehave beenbeen employedemployed toto confconfirmirm thisthis mechanism.mechanism. TheThe near-zeronear-zero ΔΔVV‡‡ valuevalue (+1.4(+1.4 cmcm33/mol/mol atat pHpH == 6.1)6.1) indicatesindicates thatthat therethere areare nono significantsignificant changeschanges inin thethe partialpartial molarmolar volumesvolumes duringduring thethe conversionconversion stepstep ofof Cbl(III)(PA)Cbl(III)(PA) toto thethe finalfinal product.product.

6.6. ConclusionsConclusions High-pressureHigh-pressure techniquestechniques forfor kinetickinetic andand memechanisticchanistic studiesstudies representrepresent aa uniqueunique andand irreplaceableirreplaceable tooltool inin thethe elucidationelucidation ofof reactionreaction mechanismsmechanisms inin solution.solution. TheThe informationinformation providedprovided byby thethe activationactivation volumevolume makesmakes itit possiblepossible toto unambiguouslyunambiguously determinedetermine thethe na-na- tureture ofof thethe underlyingunderlying reactionreaction mechanism,mechanism, whichwhich givesgives anan advantageadvantage overover thethe activationactivation

Molecules 2021, 26, 4947 21 of 24

to thoroughly explore the mechanism of the reaction of HNO with Cbl(H2O), kinetic studies were performed over a wide pH range using Piloty’s acid (PA) as the nitroxyl donor [63]. Significant differences regarding, among others, the rate-determining step of Cbl(III)(NO−) formation were found depending on the PA concentration. The results of the kinetic studies revealed the pKa of nitroxyl to be 11.4. Following the pH scale, particularly interesting observations were made in the neutral range, where the reaction product is formed remarkably fast despite the high stability of PA. This unusual behavior was explained on the basis of the formation of the Cbl(III)(PA) intermediate, which decays to nitrosylcobalamin upon deprotonation and S–N bond breaking. High-pressure techniques have been employed to confirm this mechanism. The near-zero ∆V‡ value (+1.4 cm3/mol at pH = 6.1) indicates that there are no significant changes in the partial molar volumes during the conversion step of Cbl(III)(PA) to the final product.

6. Conclusions High-pressure techniques for kinetic and mechanistic studies represent a unique and irreplaceable tool in the elucidation of reaction mechanisms in solution. The information provided by the activation volume makes it possible to unambiguously determine the nature of the underlying reaction mechanism, which gives an advantage over the activation entropy obtained by analyzing the effect of temperature. Despite the technical complexity, due in part to the relatively slow establishment of equilibrium in solution after a change in applied hydrostatic pressure, it is possible to follow the course of reactions occurring over a wide range of timescales by combining the high-pressure technique with standard, stopped-flow, and relaxation (laser flash photolysis and pulse radiolysis) techniques. This is particularly useful for studying fast inorganic reactions, which, in solution, are often applicable to biological systems. In this review, we have attempted to demonstrate the extent to which high-pressure studies have influenced the understanding of the mechanisms of NO interactions with biologically active metal centers. The redox character of these reactions, due to both the radical nature of NO and the availability of various oxidation states of metals (iron and cobalt), makes the results of pressure studies unique in providing more parameters of the complex formed than just the composition of the coordination sphere. Systematic studies extending from simple iron complexes, through Fe(II) and Fe(III) porphyrins, to protein complexes of heme (myoglobin and cytochrome P450), have demonstrated different abilities of the oxidized and reduced metal species to bind NO. Of similar importance was the need to prove the influence of ligands—in particular, the axial ligand on porphyrin complexes— in these reactions. The mechanistic information obtained from the analyses of volume profiles, and especially the position of the transition state on the volume profile, reveals new mechanistic information that was not available before. The possibility to visualize the chemical process in terms of partial molar volume changes is rather unique and opens up further theoretical work based on such volume changes. Characterization of the transition state by changing the partial molar volumes contributed to the demonstration of a wide variety of spin states, bond lengths, and electron density distributions. High-pressure studies focused on cobalt complexes—although few in number—have provided important arguments in distinguishing the biological role of Co and Fe. Complete characterizations of the intermediates in the nitrosylation reactions of both Co(II) and Co(III) porphyrins and reduced vitamin B12 are among the most important results of these studies. We are therefore fully convinced that this currently somewhat marginalized field of chemistry possessing such a powerful research tool still has much to offer.

Author Contributions: Ł.O., M.O., J.P., D.P., R.v.E. and G.S. discussed the content of the article and wrote the manuscript. All authors have read and agreed to the published version of the manuscript. Funding: The authors gratefully acknowledge ﬁnancial support from the National Science Centre in Poland (OPUS Project no. 2019/35/B/ST4/04266 and SONATA Project no. 2016/23/D/ST4/00303). Institutional Review Board Statement: Not applicable. Molecules 2021, 26, 4947 22 of 24

Informed Consent Statement: Not applicable. Conﬂicts of Interest: The authors declare no conﬂict of interest. The funders had no role in the design of the study; in the collection, analyses, or interpretation of data; in the writing of the manuscript; or in the decision to publish the results.

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