Yeast_CYT TEFKAGSAKKGATLFKTRCLQCHTVEKGGPHKVGPNLHGIFGRHSGQAEGYSYTDANIKK 60 Horse_CYT -----GDVEKGKKIFVQKCAQCHTVEKGGKHKTGPNLHGLFGRKTGQAPGFTYTDANKNK 56 Yeast_CYT NVLWDENNMSEYLTNPKKYIPGTKMAFGGLKKEKDRNDLITYLKKACE- 108 Horse_CYT GITWKEETLMEYLENPKKYIPGTKMIFAGIKKKTEREDLIAYLKKATNE 105

Figure S1. Sequence alignment of yeast and horse cyt-c (Identity~60%), green highly conserved residues. There are 40 amino acid differences in the primary sequence and 5-additional at the N-terminal of yeast. 64 residues are invariant in yeast and horse plus the CXXCH -heme binding motif and Met-80.

Supplementary Figures

pBTR1(WT)5560 bp

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A

Figure S2. Vector map of pBTR1 (unmodified) BamHI sites are marked by #

Figure S3B. Vector map of pBTR1SU (SU – Shah Ubaid-ullah) modified having incorporated restriction sites MluI and XhoI at the start and end of CYC1 gene marked by *. BamHI sites are marked by #

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Figure S3A. Cloning strategy adapted to insert restriction sites at the start and at the end of CYC1 gene. Fragment AB was amplified separately using primes Ub-1 and Ub-3 and CD was amplified using primers Ub-2 and Ub-4.Amplified fragments AB and CD were fused by denaturation and used as template in subsequent primer extension reaction using primers Ub-1 and Ub-4. Primers Ub-9 to Ub-5 carried MluI site and were used to delete the N-terminal residues of y-cyt-c.

BamHI BamHIUb-1Ub-2

Ub-3 Ub-4

MluI XhoI

Ub4Ub-8

Ub-9

Ub-7

Ub-5Ub-6

CYC1

CYC 1

A B

C D

Ub-1

Ub-4

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pBTR1SU

5544 bp

**

Figure S4. SDS PAGE of yeast iso-1-cyt c at different steps of purification. Lane 1: TCP (Total Cell Pellet), Lane 2: Supernatant (after cell lysis), Lane 3: Supernatant after NH2SO4 Cut, Lane 4: SDS VI

Marker, Lane 5: WT y-cyt-c, Lane 6: D(-5/-5), Lane 7: D(-5/-4), Lane 8: D(-5/-3), Lane 9: D(-5/-2), Lane 10: D(-5/-1).

Primer No Sequence Restriction SiteUb-1 5'- TCG TAT GTT GTG TGG AAT TGT GAG CGG ATA -3'

Ub-2 5’-CTT TAA GAA GGA GAT acg cgt ATG ACT GAA TTC AAG -3' MluI

Ub-3 5’-CTT GAA TTC AGT CAT acg cgt ATC TCC TTC TTA AAG -3' MluI

Ub-4 5'- ATA CTA TAg gat ccG ctc gag TTT ACT CAC TGG CTT -3' XhoI & BamH1

Table S1. Primers used for insertion of restriction sites in pBTR1

Table S2. Primers used for creating the N-terminal variants of yeast iso-1-cyt-c

Primers Sequence Cyt-c Variant

UB-9 5’-AAT ATA CTA acg cgt ATG GAA TTC AAG GCC GGT- 3’ Δ(-5/-5)

UB-8 5’ -AAT ATA CTA acg cgt ATG TTC AAG GCC GGT TCT- 3’ Δ(-5/-2)

UB-7 5’-AAT ATA CTA acg cgt ATG AAG GCC GGCT TCT GCT- 3’ Δ(-5/-3)

UB-6 5’-AAT ATA CTA acg cgt ATG GCC GGT TCT GCT AAG- 3’ Δ(-5/-4)

UB-5 5-’AAT ATA CTA acg cgt ATG GGT TCT GCT AAG AA- 3’ Δ(-5/-1)